Specific antibody drug conjugates (adcs) having ksp inhibitors

ABSTRACT

Specific binder-drug conjugates (ADCs) of kinesin spindle protein inhibitors, effective metabolites of these ADCs, processes for preparing these ADCs, the use of these ADCs for the treatment and/or prevention of diseases and to the use of these ADCs for preparing medicaments for treatment and/or prevention of diseases, in particular hyperproliferative and/or angiogenic disorders such as, for example, cancer diseases, are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Application No. 16/472,682,filed on Jun. 21, 2019, which is the U.S. National Stage Applicationunder 35 U.S.C. § 371 of International Application No.PCT/EP2017/083313, filed internationally on Dec. 18, 2017, which claimsthe benefit of European Application No. 16205870.5, filed on Dec. 21,2016.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on July 11, 2022, isnamed 59362-718_301_SequenceListing.xml and is 83,226 bytes in size.

INTRODUCTION AND STATE OF THE ART

The invention relates to specific binder-drug conjugates (ADCs) ofkinesin spindle protein inhibitors, to effective metabolites of theseADCs, to processes for preparing these ADCs, to the use of these ADCsfor the treatment and/or prevention of diseases and to the use of theseADCs for preparing medicaments for treatment and/or prevention ofdiseases, in particular hyperproliferative and/or angiogenic disorderssuch as, for example, cancer diseases. Such treatments can be effectedas monotherapy or else in combination with other medicaments or furthertherapeutic measures.

Cancers are the consequence of uncontrolled cell growth of the mostdiverse tissues. In many cases the new cells penetrate into existingtissue (invasive growth), or they metastasize into remote organs.Cancers occur in a wide variety of different organs and often havetissue-specific courses. The term “cancer” as a generic term thereforedescribes a large group of defined diseases of different organs, tissueand cell types. Some tumours at early stages can be removed by surgicaland radiotherapy measures. Metastasized tumours as a rule can only betreated palliatively by chemotherapeutics. The aim here is to achievethe optimum combination of an improvement in the quality of life andprolonging of life.

Conjugates of binder proteins with one or more drug molecules are known,in particular in the form of antibody drug conjugates (ADCs) in which aninternalizing antibody directed against a tumour-associated antigen iscovalently attached via a linker to a cytotoxic agent. Followingintroduction of the ADCs into the tumour cell and subsequentdissociation of the conjugate, either the cytotoxic agent itself or acytotoxic metabolite formed therefrom is released within the tumour celland can unfold its action therein directly and selectively. In thismanner, in contrast to conventional cancer chemotherapy, damage tonormal tissue is contained in significantly narrower limits [see, forexample, J. M. Lambert, Curr. Opin. Pharmacol. 5, 543-549 (2005); A. M.Wu and P. D. Senter, Nat. Biotechnol. 23, 1137-1146 (2005); P. D.Senter, Curr. Opin. Chem. Biol. 13, 235-244 (2009); L. Ducry and B.Stump, Bioconjugate Chem. 21, 5-13 (2010)]. Thus, WO2012/171020describes ADCs in which a plurality of toxophore molecules are attachedvia a polymeric linker to an antibody. As possible toxophores,WO2012/171020 mentions, among others, the substances SB 743921, SB715992 (Ispinesib), MK-0371, AZD8477, AZ3146 and ARRY-520.

The substances mentioned last are kinesin spindle protein inhibitors.Kinesin spindle protein (KSP, also known as Eg5, HsEg5, KNSL1 or KIF11)is a kinesin-like motorprotein which is essential for the bipolarmitotic spindle to function. Inhibition of KSP leads to mitotic arrestand, over a relatively long term, to apoptosis (Tao et al., Cancer Cell2005

Jul 8(1), 39-59). After the discovery of the first cell-penetrating KSPinhibitor, Monastrol, KSP inhibitors have established themselves as aclass of novel chemotherapeutics (Mayer et al., Science 286: 971-974,1999), and they are subject matter of a number of patent applications(e.g. WO2006/044825; WO2006/002236; WO2005/051922; WO2006/060737;WO03/060064; WO03/040979; and WO03/049527). However, since KSP is activeonly during a relatively short period of time during the mitosis phase,KSP inhibitors have to be present in a sufficiently high concentrationduring this phase. WO2014/151030 discloses ADCs including certain KSPinhibitors.

Further ADCs with KSP inhibitors have been disclosed in the patentapplications WO2015/096982 and WO2016/096610.

SUMMARY OF THE INVENTION

Despite various disclosures of antibody-drug conjugates, it is an objectof the present invention to provide substances which, afteradministration at a relatively low concentration, exhibit long-lastingapoptotic action and may therefore be of benefit for cancer therapy.Here, the profile of the metabolites released intracellularly from theADCs plays an important role. Frequently, the metabolites formed fromADCs are substrates of efflux pumps and/or have high cell membranepermeability. Both phenomena may contribute to a short residence timeand thus suboptimal apoptotic action in the tumour cell.

The present invention provides ADCs having a specific toxophor linkercomposition which in particular have an improved activity profile bothin association with a specific anti-CD123 antibody and with ananti-CXCRS antibody.

The antibody is preferably a humanized or chimeric monoclonal anti-CD123antibody or an anti-CXCRS antibody. Particular preference is given tothe humanized anti-CD123 antibodies TPP-8987, TPP-8988 and TPP-9476 andto the humanized or chimeric anti-CXCRS antibodies TPP-9024, TPP-9574and TPP-9580.

It has now been found that antibody-drug conjugates (ADCs) of theformula (I)

in which

n represents 1 to 8,

-   -   AK represents an anti-CD123 antibody selected from the group        consisting of TPP-8987, TPP-9476 and TPP-8988    -   or    -   AK represents an anti-CXCRS antibody, preferably selected from        the group consisting of TPP-9574, TPP-9580 and TPP-9024,    -   or    -   AK represents an antigen-binding fragment of these antibodies,

where the antibody or the antigen-binding fragment is attached via asulfur atom of a cysteine side group,

and their salts, solvates and salts of these solvates, have superiorproperties compared to the known conjugates.

Preference is given to those antibody-drug conjugates (ADCs) of theformula (I), in which n represents 4 to 8.

Preference is given to those antibody-drug conjugates (ADCs) of theformula (I), in which AK represents an anti-CD123 antibody selected fromthe group consisting of TPP-8987, TPP-9476 and TPP-8988 and anantigen-binding fragment of these antibodies; particularly preferably AKrepresents TPP-9476, and an antigen-binding fragment of this antibody.

DESCRIPTION OF THE FIGURES

FIG. 1A- FIG. 1G: Annotated sequences of preferred antibodies forbinder-drug conjugates. What are shown are the protein sequences of theheavy and light chains of the IgGs, and the VH and VL regions of theseantibodies. Below the sequences, important regions are annotated (VH andVL regions in IgGs, and the CDR regions (H-CDR1, H-CDR2, H-CDR3, L-CDR1,L-CDR2, L-CDR3)). SEQ ID NOs of annotated sequences are as follows:TPP-8987 VH (PRT)-SEQ ID NO: 1, TPP-8987 VL (PRT)-SEQ ID NO: 5, TPP-8987Heavy Chain (PRT)-SEQ ID NO: 9, TPP-8987 Light Chain (PRT)-SEQ ID NO:10, TPP-8988 VH (PRT)-SEQ ID NO: 11, TPP-8988 VL (PRT)-SEQ ID NO: 15,TPP-8988 Heavy Chain (PRT)-SEQ ID NO: 19, TPP-8988 Light Chain (PRT)-SEQID NO: 20, TPP-9024 VH (PRT)-SEQ ID NO: 21, TPP-9024 VL (PRT)-SEQ ID NO:25, TPP-9024 Heavy Chain (PRT)-SEQ ID NO: 29, TPP-9024 Light Chain(PRT)-SEQ ID NO: 30, TPP-9476 VH (PRT)-SEQ ID NO: 31, TPP-9476 VL(PRT)-SEQ ID NO: 35, TPP-9476 Heavy Chain (PRT)-SEQ ID NO: 39, TPP-9476Light Chain (PRT)-SEQ ID NO: 40, TPP-9574 VH (PRT)-SEQ ID NO: 41,TPP-9574 VL (PRT)-SEQ ID NO: 45, TPP-9574 Heavy Chain (PRT)-SEQ ID NO:49, TPP-9574 Light Chain (PRT)-SEQ ID NO: 50, TPP-9580 VH (PRT)-SEQ IDNO: 51, TPP-9580 VL (PRT)-SEQ ID NO: 55, TPP-9580 Heavy Chain (PRT)-SEQID NO: 59, and TPP-9580 Light Chain (PRT)-SEQ ID NO: 60.

FIG. 2A-FIG. 2F: Sequence listing of sequences of the preferredantibodies for binder-drug conjugates and of sequences of the targetmolecules.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides conjugates of a humanized anti-CD123 antibody ora humanized or chimeric monoclonal anti-CXCRS antibody, the drugmolecule being a kinesin spindle protein inhibitor (KSP inhibitor) whichis attached to the antibody via a linker L. Particular preference isgiven here to the humanized anti-CD123 antibodies TPP-8987, TPP-8988 andTPP-9476 and the humanized or chimeric anti-CXCRS antibodies TPP-9024,TPP-9574 and TPP-9580.

Binders

In the broadest sense, the term “binder” is understood to mean amolecule which binds to a target molecule present at a certain targetcell population to be addressed by the binder-drug conjugate. The termbinder is to be understood in its broadest meaning and also comprises,for example, lectins, proteins capable of binding to certain sugarchains, or phospholipid-binding proteins. Such binders include, forexample, high-molecular weight proteins (binding proteins), polypeptidesor peptides (binding peptides), non-peptidic (e.g. aptamers(US5,270,163) review by Keefe AD., et al., Nat. Rev. Drug Discov. 2010;9:537-550), or vitamins) and all other cell-binding molecules orsubstances. Binding proteins are, for example, antibodies and antibodyfragments or antibody mimetics, for example affibodies, adnectins,anticalins, DARPins, avimers, nanobodies (review by Gebauer M. et al.,Curr. Opinion in Chem. Biol. 2009; 13:245-255; Nuttall S.D. et al.,Curr. Opinion in Pharmacology 2008; 8:608-617). Binding peptides are,for example, ligands of a ligand/receptor pair such as, for example,VEGF of the ligand/receptor pair VEGF/KDR, such as transferrin of theligand/receptor pair transferrin/transferrin receptor orcytokine/cytokine receptor, such as TNFalpha of the ligand/receptor pairTNFalpha/TNFalpha receptor.

The binder may be a binding protein. Preferred embodiments of thebinders are an antibody, an antigen-binding antibody fragment, amultispecific antibody or an antibody mimetic.

The literature also discloses various options of covalent coupling(conjugation) of organic molecules to binders and in particularantibodies. Preference according to the invention is given to theconjugation of the toxophores to the antibody via one or more sulfuratoms of cysteine residues of the antibody and/or via one or more NHgroups of lysine residues of the antibody. However, it is also possibleto bind the toxophore to the antibody via free carboxyl groups or viasugar residues of the antibody.

A “target molecule” in the broadest sense is understood to mean amolecule which is present in the target cell population and which may bea protein (for example a receptor of a growth factor) or a non-peptidicmolecule (for example a sugar or phospholipid). It is preferably areceptor or an antigen.

The term “extracellular” target molecule describes a target molecule,attached to the cell, which is located at the outside of a cell, or thepart of a target molecule which is located at the outside of a cell,i.e. a binder may bind on an intact cell to its extracellular targetmolecule. An extracellular target molecule may be anchored in the cellmembrane or be a component of the cell membrane. The person skilled inthe art is aware of methods for identifying extracellular targetmolecules. For proteins, this may be by determining the transmembranedomain(s) and the orientation of the protein in the membrane. These dataare usually deposited in protein databases (e.g. SwissProt).

The term “cancer target molecule” describes a target molecule which ismore abundantly present on one or more cancer cell species than onnon-cancer cells of the same tissue type. Preferably, the cancer targetmolecule is selectively present on one or more cancer cell speciescompared with non-cancer cells of the same tissue type, whereselectively describes an at least two-fold enrichment on cancer cellscompared to non-cancer cells of the same tissue type (a “selectivecancer target molecule”). The use of cancer target molecules allows theselective therapy of cancer cells using the conjugates according to theinvention.

The binder can be attached to the linker via a bond. The binder can belinked by means of a heteroatom of the binder. Heteroatoms according tothe invention of the binder which can be used for attachment are sulfur(in one embodiment via a sulfhydryl group of the binder), oxygen(according to the invention by means of a carboxyl or hydroxyl group ofthe binder) and nitrogen (in one embodiment via a primary or secondaryamine group or amide group of the binder). These heteroatoms may bepresent in the natural binder or are introduced by chemical methods ormethods of molecular biology. According to the invention, the attachmentof the binder to the toxophore has only a minor effect on the bindingactivity of the binder with respect to the target molecule. In apreferred embodiment, the linkage has no effect on the binding activityof the binder with respect to the target molecule.

In accordance with the present invention, the term “antibody” is to beunderstood in its broadest meaning and comprises immunoglobulinmolecules, for example intact or modified monoclonal antibodies,polyclonal antibodies or multispecific antibodies (e.g. bispecificantibodies). An immunoglobulin molecule preferably comprises a moleculehaving four polypeptide chains, two heavy chains (H chains) and twolight chains (L chains) which are typically linked by disulfide bridges.Each heavy chain comprises a variable domain of the heavy chain(abbreviated VH) and a constant domain of the heavy chain. The constantdomain of the heavy chain may, for example, comprise three domains CH1,CH2 and CH3. Each light chain comprises a variable domain (abbreviatedVL) and a constant domain. The constant domain of the light chaincomprises a domain (abbreviated CL). The VH and VL domains may besubdivided further into regions having hypervariability, also referredto as complementarity determining regions (abbreviated CDR) and regionshaving low sequence variability (framework region, abbreviated FR).Typically, each VH and VL region is composed of three CDRs and up tofour FRs. For example from the amino terminus to the carboxy terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. An antibodymay be obtained from any suitable species, e.g. rabbit, llama, camel,mouse or rat. In one embodiment, the antibody is of human or murineorigin. An antibody may, for example, be human, humanized or chimeric.

The term “monoclonal” antibody refers to antibodies obtained from apopulation of substantially homogeneous antibodies, i.e. individualantibodies of the population are identical except for naturallyoccurring mutations, of which there may be a small number. Monoclonalantibodies recognize a single antigenic binding site with highspecificity. The term monoclonal antibody does not refer to a particularpreparation process.

The term “intact” antibody refers to antibodies comprising both anantigen-binding domain and the constant domain of the light and heavychain. The constant domain may be a naturally occurring domain or avariant thereof having a number of modified amino acid positions, andmay also be aglycosylated.

The term “modified intact” antibody refers to intact antibodies fusedvia their amino terminus or carboxy terminus by means of a covalent bond(e.g. a peptide bond) with a further polypeptide or protein notoriginating from an antibody. Furthermore, antibodies may be modifiedsuch that, at defined positions, reactive cysteines are introduced tofacilitate coupling to a toxophore (see Junutula et al. Nat Biotechnol.2008 Aug;26(8):925-32).

“Amino acid modification” or “mutation” here means an amino acidsubstitution, insertion and/or deletion in a polypeptide sequence. Thepreferred amino acid modification here is a substitution. “Amino acidsubstitution” or “substitution” here means an exchange of an amino acidat a given position in a protein sequence for another amino acid. Forexample, the substitution Y5OW describes a variant of a parentpolypeptide in which the tyrosine at position 50 has been exchanged fora tryptophan. A “variant” of a polypeptide describes a polypeptidehaving an amino acid sequence substantially identical to a referencepolypeptide, typically a native or “parent” polypeptide. The polypeptidevariant may have one or more amino acid exchanges, deletions and/orinsertions at particular positions in the native amino acid sequence.

The term “human” antibody refers to antibodies which can be obtainedfrom a human or which are synthetic human antibodies. A “synthetic”human antibody is an antibody which is partially or entirely obtainablein silico from synthetic sequences based on the analysis of humanantibody sequences. A human antibody can be encoded, for example, by anucleic acid isolated from a library of antibody sequences of humanorigin. An example of such an antibody can be found in Soderlind et al.,Nature Biotech. 2000, 18:853-856. Such “human” and “synthetic”antibodies also include aglycosylated variants which have been producedeither by deglycosylation by PNGaseF or by mutation of N297 (Kabatnumbering) of the heavy chain to any other amino acid.

The term “humanized” or “chimeric” antibody describes antibodiesconsisting of a non-human and a human portion of the sequence. In theseantibodies, part of the sequences of the human immunoglobulin(recipient) is replaced by sequence portions of a non-humanimmunoglobulin (donor). In many cases, the donor is a murineimmunoglobulin. In the case of humanized antibodies, amino acids of theCDR of the recipient are replaced by amino acids of the donor.Sometimes, amino acids of the framework, too, are replaced bycorresponding amino acids of the donor. In some cases the humanizedantibody contains amino acids present neither in the recepient nor inthe donor, which were introduced during the optimization of theantibody. In the case of chimeric antibodies, the variable domains ofthe donor immunoglobulin are fused with the constant regions of a humanantibody. Such “humanized” and “chimeric” antibodies also includeaglycosylated variants which have been produced either bydeglycosylation by PNGaseF or by mutation of N297 (Kabat numbering) ofthe heavy chain to any other amino acid.

The term complementarity determining region (CDR) as used herein refersto those amino acids of a variable antibody domain which are requiredfor binding to the antigen. Typically, each variable region has threeCDR regions referred to as CDR1, CDR2 and CDR3. Each CDR region mayembrace amino acids according to the definition of Kabat and/or aminoacids of a hypervariable loop defined according to Chotia. Thedefinition according to Kabat comprises, for example, the region fromabout amino acid position 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) ofthe variable light chain/domain (VL) and 31-35 (CDR1), 50-65 (CDR2) and95-102 (CDR3) of the variable heavy chain/domain (VH) (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, MD. (1991)). Thedefinition according to Chotia comprises, for example, the region fromabout amino acid position 26-32 (CDR1), 50-52 (CDR2) and 91-96 (CDR3) ofthe variable light chain (VL) and 26-32 (CDR1), 53-55 (CDR2) and 96-101(CDR3) of the variable heavy chain (VH) (Chothia and Lesk; J Mol Biol196: 901-917 (1987)). In some cases, a CDR may comprise amino acids froma CDR region defined according to Kabat and Chotia.

Depending on the amino acid sequence of the constant domain of the heavychain, antibodies may be categorized into different classes. There arefive main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, andseveral of these can be divided into further subclasses. (Isotypes),e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The constant domains of theheavy chain, which correspond to the different classes, are referred toas [alpha/α], [delta/δ], [epsilon/ε], [gamma/γ] and [my/μ]. Both thethree-dimensional structure and the subunit structure of antibodies areknown.

The term “functional fragment” or “antigen-binding antibody fragment” ofan antibody/immunoglobulin is defined as a fragment of anantibody/immunoglobulin (e.g. the variable domains of an IgG) whichstill comprise the antigen binding domains of theantibody/immunoglobulin. The “antigen binding domain” of an antibodytypically comprises one or more hypervariable regions of an antibody,for example the CDR, CDR2 and/or CDR3 region. However, the “framework”or “skeleton” region of an antibody may also play a role during bindingof the antibody to the antigen. The framework region forms the skeletonof the CDRs. Preferably, the antigen binding domain comprises at leastamino acids 4 to 103 of the variable light chain and amino acids 5 to109 of the variable heavy chain, more preferably amino acids 3 to 107 ofthe variable light chain and 4 to 111 of the variable heavy chain,especially preferably the complete variable light and heavy chains, i.e.amino acids 1-109 of the VL and 1 to 113 of the VH (numbering accordingto WO97/08320).

“Functional fragments” or “antigen-binding antibody fragments” of theinvention encompass, non-conclusively, Fab, Fab′, F(ab′)₂ and Fvfragments, diabodies, Single Domain Antibodies (DAbs), linearantibodies, individual chains of antibodies (single-chain Fv,abbreviated to scFv); and multispecific antibodies, such as bi- andtri-specific antibodies, for example, formed from antibody fragments C.A. K Borrebaeck, editor (1995) Antibody Engineering (Breakthroughs inMolecular Biology), Oxford University

Press; R. Kontermann & S. Duebel, editors (2001) Antibody Engineering(Springer Laboratory Manual), Springer Verlag. Antibodies other than“multispecific” or “multifunctional” antibodies are those havingidentical binding sites. Multispecific antibodies may be specific fordifferent epitopes of an antigen or may be specific for epitopes of morethan one antigen (see, for example, WO 93/17715; WO 92/08802; WO91/00360; WO 92/05793; Tutt, et al., 1991, J. Immunol. 14760 69; U. S.Pat. Nos. 4,474,893; 4,7 14,68 1 ; 4,925,648; 5,573,920; 5,601,8 19; orKostelny et al., 1992, J. Immunol. 148 1547 1553). An F(ab′)₂ or Fabmolecule may be constructed such that the number of intermoleculardisulfide interactions occurring between the Ch1 and the CL domains canbe reduced or else completely prevented.

“Epitopes” refer to protein determinants capable of binding specificallyto an immunoglobulin or T cell receptors. Epitopic determinants usuallyconsist of chemically active surface groups of molecules such as aminoacids or sugar side chains or combinations thereof, and usually havespecific 3-dimensional structural properties and also specific chargeproperties.

“Functional fragments” or “antigen-binding antibody fragments” may befused with another polypeptide or protein, not originating from anantibody, via the amino terminus or carboxyl terminus thereof, by meansof a covalent bond (e.g. a peptide linkage). Furthermore, antibodies andantigen-binding fragments may be modified by introducing reactivecysteines at defined locations, in order to facilitate coupling to atoxophore (see Junutula et al. Nat Biotechnol. 2008 Aug; 26(8):925-32).

Polyclonal antibodies can be prepared by methods known to a person ofordinary skill in the art. Monoclonal antibodies may be prepared bymethods known to a person of ordinary skill in the art (Köhler andMilstein, Nature, 256, 495-497, 1975). Human and humanized monoclonalantibodies may be prepared by methods known to a person of ordinaryskill in the art (Olsson et al., Meth Enzymol. 92, 3-16 or Cabilly et alU.S. Pat. No. 4,816,567 or Boss et al U.S. Pat. No. 4,816,397).

A person of ordinary skill in the art is aware of diverse methods forpreparing human antibodies and fragments thereof, such as, for example,by means of transgenic mice (N Lonberg and D Huszar, Int Rev Immunol.1995; 13(1):65-93) or Phage Display Technologien (Clackson et al.,Nature. 1991 Aug 15;352(6336):624-8). Antibodies of the invention may beobtained from recombinant antibody libraries consisting for example onthe amino acid sequences of a multiplicity of antibodies compiled from alarge number of healthy volunteers. Antibodies may also be produced bymeans of known recombinant DNA technologies. The nucleic acid sequenceof an antibody can be obtained by routine sequencing or is availablefrom publically accessible databases.

An “isolated” antibody or binder has been purified to remove otherconstituents of the cell. Contaminating constituents of a cell which mayinterfere with a diagnostic or therapeutic use are, for example,enzymes, hormones, or other peptidic or non-peptidic constituents of acell. A preferred antibody or binder is one which has been purified toan extent of more than 95% by weight, relative to the antibody or binder(determined for example by Lowry method, UV-Vis spectroscopy or by SDScapillary gel electrophoresis). Moreover an antibody which has beenpurified to such an extent that it is possible to determine at least 15amino acids of the amino terminus or of an internal amino acid sequence,or which has been purified to homogeneity, the homogeneity beingdetermined by SDS-PAGE under reducing or non-reducing conditions(detection may be determined by means of Coomassie Blue staining orpreferably by silver coloration). However, an antibody is normallyprepared by one or more purification steps.

The term “specific binding” or “binds specifically” refers to anantibody or binder which binds to a predetermined antigen/targetmolecule. Specific binding of an antibody or binder typically describesan antibody or binder having an affinity of at least 10⁻⁷ M (as Kdvalue; i.e. preferably those with Kd values smaller than 10⁻⁷ M), withthe antibody or binder having an at least two times higher affinity forthe predetermined antigen/target molecule than for a non-specificantigen/target molecule (e.g. bovine serum albumin, or casein) which isnot the predetermined antigen/target molecule or a closely relatedantigen/target molecule. Specific binding of an antibody or binder doesnot exclude the antibody or binder binding to a plurality ofantigens/target molecules (e.g. orthologs of different species). Theantibodies preferably have an affinity of at least 10⁻⁷ M (as Kd value;in other words preferably those with smaller Kd values than 10⁻⁷ M),preferably of at least 10⁻⁸ M, more preferably in the range from 10⁻⁹ Mto 10⁻¹¹ M. The Kd values may be determined, for example, by means ofsurface plasmon resonance spectroscopy.

The antibody-drug conjugates of the invention likewise exhibitaffinities in these ranges. The affinity is preferably not substantiallyaffected by the conjugation of the drugs (in general, the affinity isreduced by less than one order of magnitude, in other words, forexample, at most from 10⁻⁸ M to 10⁻⁷ M).

The antibodies used in accordance with the invention are also notablepreferably for a high selectivity. A high selectivity exists when theantibody of the invention exhibits an affinity for the target proteinwhich is better by a factor of at least 2, preferably by a factor of 5or more preferably by a factor of 10, than for an independent otherantigen, e.g. human serum albumin (the affinity may be determined, forexample, by means of surface plasmon resonance spectroscopy).

Furthermore, the antibodies of the invention that are used arepreferably cross-reactive. In order to be able to facilitate and betterinterpret preclinical studies, for example toxicological or activitystudies (e.g. in xenograft mice), it is advantageous if the antibodyused in accordance with the invention not only binds the human targetprotein but also binds the species target protein in the species usedfor the studies. In one embodiment the antibody used in accordance withthe invention, in addition to the human target protein, iscross-reactive to the target protein of at least one further species.For toxicological and activity studies it is preferred to use species ofthe families of rodents, dogs and non-human primates. Preferred rodentspecies are mouse and rat. Preferred non-human primates are rhesusmonkeys, chimpanzees and long-tailed macaques.

In one embodiment the antibody used in accordance with the invention, inaddition to the human target protein, is cross-reactive to the targetprotein of at least one further species selected from the group ofspecies consisting of mouse, rat and long-tailed macaque

(Macaca fascicularis). Especially preferred are antibodies used inaccordance with the invention which in addition to the human targetprotein are at least cross-reactive to the mouse target protein.Preference is given to cross-reactive antibodies whose affinity for thetarget protein of the further non-human species differs by a factor ofnot more than 50, more particularly by a factor of not more than ten,from the affinity for the human target protein.

Antibodies Directed against a Cancer Target Molecule

The target molecule towards which the binder, for example an antibody oran antigen-binding fragment thereof, is directed is preferably a cancertarget molecule. The term “cancer target molecule” describes a targetmolecule which is more abundantly present on one or more cancer cellspecies than on non-cancer cells of the same tissue type. Preferably,the cancer target molecule is selectively present on one or more cancercell species compared with non-cancer cells of the same tissue type,where selectively describes an at least two-fold enrichment on cancercells compared to non-cancer cells of the same tissue type (a “selectivecancer target molecule”). The use of cancer target molecules allows theselective therapy of cancer cells using the conjugates according to theinvention.

Antibodies which are specific against an antigen, for example cancercell antigen, can be prepared by a person of ordinary skill in the artby means of methods with which he or she is familiar (such asrecombinant expression, for example) or may be acquired commercially (asfor example from Merck KGaA, Germany). Examples of known commerciallyavailable antibodies in cancer therapy are Erbitux® (cetuximab, MerckKGaA), Avastin® (bevacizumab, Roche) and Herceptin® (trastuzumab,Genentech). Trastuzumab is a recombinant humanized monoclonal antibodyof the IgG1kappa type which in a cell-based assay (Kd=5 nM) binds theextracellular domains of the human epidermal growth receptor with highaffinity. The antibody is produced recombinantly in CHO cells. All theseantibodies can also be produced as aglycosylated variants of theseantibodies, either by deglycosylation by means of PNGase F or bymutation of N297 (Kabat numbering) of the heavy chain to any amino acid.

In the present invention, the cancer target molecules are

(1) the receptor protein CXCRS (CD185; SwissProt: P32302; NCBI Gene ID643, NCBI Reference Sequence: NP_001707.1; SEQ ID NO: 61)

(2) the surface receptor CD123 (IL3RA; NCBI Gene ID: 3563; NCBIReference Sequence: NP_002174.1; Swiss-Prot: P26951; SEQ ID NO: 62)

In particularly preferred subject of the invention, the binder bindsspecifically to an extracellular cancer target molecule selected fromthe group consisting of of the cancer target molecules CXCR5 and CD123.In a preferred embodiment, the binder, after binding to itsextracellular target molecule on the target cell, is internalized by thetarget cell through the binding. This causes the binder-drug conjugate,which may be an immuno-conjugate or an ADC, to be taken up by the targetcell. The binder is then processed, preferably intracellularly, withpreference lysosomally.

In one embodiment the binder is a binding protein. In a preferredembodiment the binder is an antibody, an antigen-binding antibodyfragment, a multispecific antibody or an antibody mimetic.

Preferred antibody mimetics are affibodies, adnectins, anticalins,DARPins, avimers, or nanobodies. Preferred multispecific antibodies arebispecific and trispecific antibodies.

In a preferred embodiment the binder is an antibody or anantigen-binding antibody fragment, more preferably an isolated antibodyor an isolated antigen-binding antibody fragment.

Preferred antigen-binding antibody fragments are Fab, Fab′, F(ab′)2 andFv fragments, diabodies, DAbs, linear antibodies and scFv. Particularlypreferred are Fab, diabodies and scFv.

In a particularly preferred embodiment the binder is an antibody.Particularly preferred are monoclonal antibodies or antigen-bindingantibody fragments thereof. Further particularly preferred are human,humanized or chimeric antibodies or antigen-binding antibody fragmentsthereof.

Antibodies or antigen-binding antibody fragments which bind cancertarget molecules may be prepared by a person of ordinary skill in theart using known processes, such as, for example, chemical synthesis orrecombinant expression. Binders for cancer target molecules may beacquired commercially or may be prepared by a person of ordinary skillin the art using known processes, such as, for example, chemicalsynthesis or recombinant expression. Further processes for preparingantibodies or antigen-binding antibody fragments are described in WO2007/070538 (see page 22 “Antibodies”). The person skilled in the artknows how processes such as phage display libraries (e.g. MorphosysHuCAL Gold) can be compiled and used for discovering antibodies orantigen-binding antibody fragments (see WO 2007/070538, page 24 ff andAK Example 1 on page 70, AK Example 2 on page 72). Further processes forpreparing antibodies that use DNA libraries from B cells are describedfor example on page 26 (WO 2007/070538). Processes for humanizingantibodies are described on page 30-32 of WO2007070538 and in detail inQueen, et al., Pros. Natl. Acad. Sci. USA 8610029-10033,1989 or in WO90/0786. Furthermore, processes for recombinant expression of proteinsin general and of antibodies in particular are known to the personskilled in the art (see, for example, in Berger and Kimmel (Guide toMolecular Cloning Techniques, Methods in Enzymology, Vol. 152, AcademicPress, Inc.); Sambrook, et al., (Molecular Cloning A Laboratory Manual,(Second Edition, Cold Spring Harbor Laboratory Press; Cold SpringHarbor, N.Y.; 1989) Vol. 1-3); Current Protocols in Molecular Biology,(F. M. Ausabel et al. [Eds.], Current Protocols, Green PublishingAssociates, Inc./John Wiley & Sons, Inc.); Harlow et al., (MonoclonalAntibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press(19881, Paul [Ed.]); Fundamental Immunology, (Lippincott Williams &Wilkins (1998)); and Harlow, et al., (Using Antibodies A LaboratoryManual, Cold Spring Harbor Laboratory Press (1998)). The person skilledin the art knows the corresponding vectors, promoters and signalpeptides which are necessary for the expression of a protein/antibody.Commonplace processes are also described in WO 2007/070538 on pages41-45. Processes for preparing an IgG1 antibody are described forexample in WO 2007/070538 in Example 6 on page 74 ff. Processes whichallow the determination of the internalization of an antibody afterbinding to its antigen are known to the skilled person and are describedfor example in WO 2007/070538 on page 80. The person skilled in the artis able to use the processes described in WO 2007/070538 that have beenused for preparing carboanhydrase IX (Mn) antibodies in analogy for thepreparation of antibodies with different target molecule specificity.

Bacterial Expression

The person skilled in the art is aware of the way in which antibodies,antigen-binding fragments thereof or variants thereof can be producedwith the aid of bacterial expression.

Suitable expression vectors for bacterial expression of desired proteinsare constructed by insertion of a DNA sequence which encodes the desiredprotein within the functional reading frame together with suitabletranslation initiation and translation termination signals and with afunctional promoter. The vector comprises one or more phenotypicallyselectable markers and a replication origin in order to enable theretention of the vector and, if desired, the amplification thereofwithin the host. Suitable prokaryotic hosts for transformation includebut are not limited to E. coli, Bacillus subtilis, Salmonellatyphimurium and various species from the genus Pseudomonas,Streptomyces, and Staphylococcus. Bacterial vectors may be based, forexample, on bacteriophages, plasmids, or phagemids. These vectors maycontain selectable markers and a bacterial replication origin, which arederived from commercially available plasmids. Many commerciallyavailable plasmids typically contain elements of the well-known cloningvector pBR322 (ATCC 37017). In bacterial systems, a number ofadvantageous expression vectors can be selected on the basis of theintended use of the protein to be expressed.

After transformation of a suitable host strain and growth of the hoststrain to an appropriate cell density, the selected promoter isde-reprimed/induced by suitable means (for example a change intemperature or chemical induction), and the cells are cultivated for anadditional period. The cells are typically harvested by centrifugationand if necessary digested in a physical manner or by chemical means, andthe resulting raw extract is retained for further purification.

Therefore, a further embodiment of the present invention is anexpression vector comprising a nucleic acid which encodes a novelantibody of the present invention.

Antibodies of the present invention or antigen-binding fragments thereofinclude naturally purified products, products which originate fromchemical syntheses, and products which are produced by recombinanttechnologies in prokaryotic hosts, for example E. coli, Bacillussubtilis, Salmonella typhimurium and various species from the genusPseudomonas, Streptomyces, and Staphylococcus, preferably E. coli.

Mammalian Cell Expression

The person skilled in the art is aware of the way in which antibodies,antigen-binding fragments thereof or variants thereof can be producedwith the aid of mammalian cell expression.

Preferred regulatory sequences for expression in mammalian cell hostsinclude viral elements which lead to high expression in mammalian cells,such as promoters and/or expression amplifiers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), simian virus40 (SV40) (such as the SV40 promoter/enhancer), from adenovirus, (forexample the adenovirus major late promoter (AdMLP)) and from polyoma.The expression of the antibodies may be constitutive or regulated (forexample induced by addition or removal of small molecule inductors suchas tetracycline in combination with the Tet system).

For further description of viral regulatory elements and sequencesthereof, reference is made, for example, to U.S. Pat. No. 5,168,062 byStinski, U.S. Pat. No. 4,510,245 by Bell et al. and U.S. Pat. No.4,968,615 by Schaffner et al. The recombinant expression vectors maylikewise include a replication origin and selectable markers (see, forexample, U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). Suitableselectable markers include genes which impart resistance to substancessuch as G418, puromycin, hygromycin, blasticidin, zeocin/bleomycin, ormethotrexate, or selectable markers which lead to auxotrophy of a hostcell, such as glutamine synthetase (Bebbington et al., Biotechnology (NY). 1992 Feb;10(2):169-75), when the vector has been introduced into thecell.

For example, the dihydrofolate reductase (DHFR) gene imparts resistanceto methotrexate, the neo gene imparts resistance to G418, the bsd genefrom Aspergillus terreus imparts resistance to blasticidin, puromycinN-acetyltransferase imparts resistance to puromycin, the Sh ble geneproduct imparts resistance to zeocin, and resistance to hygromycin isimparted by the E. coli hygromycin resistance gene (hyg or hph).Selectable markers such as DHFR or glutamine synthetase are also helpfulfor amplification techniques in conjunction with MTX and MSX.

The transfection of an expression vector into a host cell can beexecuted with the aid of standard techniques, including byelectroporation, nucleofection, calcium phosphate precipitation,lipofection, polycation-based transfection such as polyethyleneimine(PEI)-based transfection and DEAE-dextran transfection.

Suitable mammalian host cells for the expression of antibodies,antigen-binding fragments thereof, or variants thereof include Chinesehamster ovary (CHO) cells such as CHO-K1, CHO-S, CHO-K1SV [includingDHFR-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220 and Urlaub et al., Cell. 1983 June;33(2):405-12,used with a DHFR-selectable marker, as described in R. J. Kaufman and P.A. Sharp (1982) Mol. Biol. 159:601-621, and other knockout cells, asdetailed in Fan et al., Biotechnol Bioeng. 2012 April;109(4):1007-15),NSO myeloma cells, COS cells, HEK293 cells, HKB11 cells, BHK21 cells,CAP cells, EB66 cells, and SP2 cells.

The expression of antibodies, antigen-binding fragments thereof, orvariants thereof can also be effected in a transient or semi-stablemanner in expression systems such as HEK293, HEK293T, HEK293-EBNA,HEK293E, HEK293-6E, HEK293 Freestyle, HKB11, Expi293F, 293EBNALT75, CHOFreestyle, CHO-S, CHO-K1, CHO-K1SV, CHOEBNALT85, CHOS-XE, CHO-3E7 orCAP-T cells (for example like Durocher et al., Nucleic Acids Res. 2002Jan 15;30(2):E9)

In some embodiments, the expression vector is constructed in such a waythat the protein to be expressed is secreted into the cell culturemedium in which the host cells are growing. The antibodies, theantigen-binding fragments thereof, or the variants thereof can beobtained from the cell culture medium with the aid of proteinpurification methods known to those skilled in the art.

Purification

The antibodies, the antigen-binding fragments thereof, or the variantsthereof can be obtained and purified from recombinant cell cultures withthe aid of well-known methods, examples of which include ammoniumsulfate or ethanol precipitation, acid extraction, protein Achromatography, protein G chromatography, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography (HIC), affinity chromatography, hydroxyapatitechromatography and lectin chromatography. High-performance liquidchromatography (“HPLC”) can likewise be employed for purification. See,for example, Colligan, Current Protocols in Immunology, or CurrentProtocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001),e.g., Chapters 1, 4, 6, 8, 9, 10.

Antibodies of the present invention or antigen-binding fragmentsthereof, or variants thereof include naturally purified products,products from chemical synthesis methods and products which are producedwith the aid of recombinant techniques in prokaryotic or eukaryotic hostcells. Eukaryotic hosts include, for example, yeast cells, higher plantcells, insect cells and mammalian cells. Depending on the host cellchosen for the recombinant expression, the protein expressed may be inglycosylated or non-glycosylated form.

In a preferred embodiment, the antibody is purified (1) to an extent ofmore than 95% by weight, measured, for example, by the Lowry method, byUV-vis spectroscopy or by SDS capillary gel electrophoresis (for examplewith a Caliper LabChip GXII, GX 90 or Biorad Bioanalyzer instrument),and in more preferred embodiments more than 99% by weight, (2) to adegree suitable for determination of at least 15 residues of theN-terminal or internal amino acid sequence, or (3) to homogeneitydetermined by SDS-PAGE under reducing or non-reducing conditions withthe aid of Coomassie blue or preferably silver staining.

Usually, an isolated antibody is obtained with the aid of at least oneprotein purification step.

Anti-CD123 Antibodies

According to the invention, it is possible to use anti-CD123 antibodies.

The expression “anti-CD123 antibody” or “an antibody which bindsspecifically to CD123” relates to an antibody which binds the cancertarget molecule CD123 (NCBI Reference sequence: NP_002174.1; SEQ ID NO:62), preferably with an affinity sufficient for a diagnostic and/ortherapeutic application. In particular embodiments, the antibody bindsCD123 with a dissociation constant (K_(D)) of ≤1μM, ≤100 nM, ≤10 nM, ≤1nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM.

Sun et al. (Sun et al., 1996, Blood 87(1)83-92) describe the generationand properties of the monoclonal antibody 7G3, which binds theN-terminal domain of IL-3Rα, CD123. U.S. Pat. No. 6,177,078 (Lopez)relates to the anti-CD123 antibody 7G3. A chimeric variant of thisantibody (CSL360) is described in WO 2009/070844, and a humanizedversion (CSL362) in WO 2012/021934. The sequence of the 7G3 antibody isdisclosed in EP2426148. This sequence constitutes the starting point forthe humanized antibodies obtained by CDR grafting.

An antibody which, after cell surface antigen binding, is internalizedparticularly well is the anti-CD123 antibody 12F1 disclosed by Kuo etal. (Kuo et al., 2009, Bioconjug Chem. 20(10):1975-82). The antibody12F1 binds with higher affinity to CD123 than the antibody 7G3 and,after cell surface antigen binding, is internalized markedly faster than7G3. Bispecific scFv immunofusion proteins based on 12F1 are disclosedin WO 2013/173820. Antibody TPP-6013 is a chimeric variant of 12F1.

The invention relates in particular to conjugates with antibodies orantigen-binding antibody fragments thereof or variants thereof derivedfrom the antibodies 7G3 (Sun et al., 1996, Blood 87(1):83-92) and 12F1(Kuo et al., 2009, Bioconjug Chem. 20(10):1975-82) originating from themouse, or to conjugates with antibodies or antigen-binding antibodyfragments thereof or variants thereof derived from the antibody 12F1(Kuo et al., 2009, Bioconjug Chem. 20(10):1975-82) originating from themouse.

Particular preference is given in the context of the present inventionto the anti-CD123 antibodies TPP-9476, TPP-8988 and TPP-8987.

Anti-CXCR5 Antibodies

According to the invention, it is possible to use anti-CXCR5 antibodies.

The expression “anti-CXCR5 antibody” or “an antibody which bindsspecifically to CXCR5” relates to an antibody which binds the cancertarget molecule CXCR5 (NCBI Reference Sequence: NP_001707.1; SEQ ID NO:61), preferably with an affinity sufficient for a diagnostic and/ortherapeutic application. In particular embodiments, the antibody bindsCXCR5 with a dissociation constant (K_(D)) of ≤1μM, ≤100 nM, ≤10 nM, ≤1nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM.

Examples of antibodies and antigen-binding fragments which bind to CXCR5are known to those skilled in the art and are described, for example, inEP2195023.

The hybridoma cells for the rat antibody RF8B2 (ACC2153) were purchasedfrom DSMZ and the sequence of the antibody was identified by standardmethods. TPP-9024 a chimeric variant of this antibody with a pointmutation at position 67 (S67F) was prepared. Furthermore, the ratantibody sequence constituted the starting point for the humanizedantibodies obtained by CDR grafting into human framework.

These antibodies and antigen-binding fragments can be used in thecontext of this invention.

Particular preference is given in the context of the present inventionto the humanized anti-CXCR5 antibodies TPP-9574, TPP-9580 and thechimeric antibody TPP-9024.

Preferred Antibodies and Antigen-Binding Antibody Fragments forBinder-Drug Conjugates according to the Invention

In this application, in the context of the binder-drug conjugates,reference is made to the following preferred antibodies as shown in thefollowing table: anti-CD123 antibodies TPP-8987, TPP-8988 and TPP-9476and the anti-CXCR5 antibodies TPP-9024, TPP-9574 and TPP-9580.

Table: Protein sequences of the preferred antibodies: SEQ SEQ ID NO: IDNO: SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ IgG IgG Antibody ID NO: ID NO: IDNO: ID NO: ID NO: ID NO: ID NO: ID NO: heavy light TPP-XXX Antigen VHH-CDR1 H-CDR2 H-CDR3 VL L-CDR1 L-CDR2 L-CDR3 chain chain TPP-8987 CD123 1  2  3  4  5  6  7  8  9 10 TPP-8988 CD123 11 12 13 14 15 16 17 18 1920 TPP-9024 CXCR5 21 22 23 24 25 26 27 28 29 30 TPP-9476 CD123 31 32 3334 35 36 37 38 39 40 TPP-9574 CXCR5 41 42 43 44 45 46 47 48 49 50TPP-9580 CXCR5 51 52 53 54 55 56 57 58 59 60

TPP-8987, TPP-8988, TPP-9476, TPP-9024, TPP-9574 and TPP-9580 areantibodies comprising one or more of the CDR sequences specified in theabove table (H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, L-CDR3) in thevariable region of the heavy chain (VH) or the variable region of thelight chain (VL). Preferably, the antibodies comprise the specifiedvariable region of the heavy chain (VH) and/or the variable region ofthe light chain (VL). Preferably, the antibodies comprise the specifiedregion of the heavy chain (IgG heavy chain) and/or the specified regionof the light chain (IgG light chain).

TPP-8987 is an anti-CD123 antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 2, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 3 and the variable CDR3sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 4, and avariable region of the light chain (VL) comprising the variable CDR1sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 6, thevariable CDR2 sequence of the light chain (L-CDR2), as shown by SEQ IDNO: 7 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 8.

TPP-8988 is an anti-CD123 antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 12, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 13 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 14,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 16,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 17 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 18.

TPP-9024 is an anti-CXCRS antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 22, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 23 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 24,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 26,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 27 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 28.

TPP-9476 is an anti-CD123 antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 32, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 33 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 34,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 36,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 37 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 38.

TPP-9574 is an anti-CXCRS antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 42, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 43 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 44,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 46,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 47 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 48.

TPP-9580 is an anti-CXCRS antibody comprising a variable region of theheavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 52, the variable CDR2 sequence ofthe heavy chain (H-CDR2), as shown by SEQ ID NO: 53 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 54,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L-CDR1), as shown by SEQ ID NO: 56,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 57 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 58.

TPP-8987 is an anti-CD123 antibody comprising preferably a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 1 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 5.

TPP-8988 is an anti-CD123 antibody comprising preferably a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 11 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 15.

TPP-9024 is an anti-CXCRS antibody comprising preferably a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 21 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 25.

TPP-9476 is an anti-CD123 antibody comprising preferably a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 31 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 35.

TPP-9574 is an anti-CXCRS antibody comprising preferably a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 41 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 45.

TPP-9580 is an anti-CXCRS antibody comprising preferably a variableregion of the heavy chain (VH) as shown in SEQ ID NO: 51 and a variableregion of the light chain (VL) as shown in SEQ ID NO: 55.

TPP-8987 is an anti-CD123 antibody comprising preferably a region of theheavy chain as shown in SEQ ID NO: 9 and a region of the light chain asshown in SEQ ID NO: 10.

TPP-8988 is an anti-CD123 antibody comprising preferably a region of theheavy chain as shown in SEQ ID NO: 19 and a region of the light chain asshown in SEQ ID NO: 20.

TPP-9024 is an anti-CXCRS antibody comprising preferably a region of theheavy chain as shown in SEQ ID NO: 29 and a region of the light chain asshown in SEQ ID NO: 30.

TPP-9476 is an anti-CD123 antibody comprising preferably a region of theheavy chain as shown in SEQ ID NO: 39 and a region of the light chain asshown in SEQ ID NO: 40.

TPP-9574 is an anti-CXCRS antibody comprising preferably a region of theheavy chain as shown in SEQ ID NO: 49 and a region of the light chain asshown in SEQ ID NO: 50.

TPP-9580 is an anti-CXCRS antibody comprising preferably a region of theheavy chain as shown in SEQ ID NO: 59 and a region of the light chain asshown in SEQ ID NO: 60.

Therapeutic use

The hyper-proliferative diseases, for the treatment of which thecompounds according to the invention may be employed, include inparticular the group of cancer and tumour diseases. In the context ofthe present invention, these are understood to mean especially thefollowing diseases, but without any limitation thereto: mammarycarcinomas and mammary tumours (mammary carcinomas including ductal andlobular forms, also in situ), tumours of the respiratory tract(small-cell and non-small cell carcinoma, bronchial carcinoma), cerebraltumours (e.g. of the brain stem and of the hypothalamus, astrocytoma,ependymoma, glioblastoma, glioma, medulloblastoma, meningioma andneuro-ectodermal and pineal tumours), tumours of the digestive organs(carcinomas of the oesophagus, stomach, gall bladder, small intestine,large intestine, rectum and anal carcinomas), liver tumours (inter aliahepatocellular carcinoma, cholangiocarcinoma and mixed hepatocellularcholangiocarcinoma), tumours of the head and neck region (larynx,hypopharynx, nasopharynx, oropharynx, lips and oral cavity carcinomas,oral melanomas), skin tumours (basaliomas, spinaliomas, squamous cellcarcinomas, Kaposi's sarcoma, malignant melanoma, non-melanomatous skincancer, Merkel cell skin cancer, mast cell tumours), tumours ofconnective tissue (inter alia soft tissue sarcomas, osteosarcomas,malignant fibrous histiocytomas, chondrosarcomas, fibrosarcomas,haemangiosarcomas, leiomyosarcomas, liposarcomas, lymphosarcomas andrhabdomyosarcomas), tumours of the eyes (inter alia intraocular melanomaand retinoblastoma), tumours of the endocrine and exocrine glands (e.g.of the thyroid and parathyroid glands, pancreas and salivary glandcarcinomas, adenocarcinomas), tumours of the urinary tract (tumours ofthe bladder, penis, kidney, renal pelvis and ureter) and tumours of thereproductive organs (carcinomas of the endometrium, cervix, ovary,vagina, vulva and uterus in women and carcinomas of the prostate andtestes in men). These also include proliferative diseases of the blood,the lymph system and the spinal cord, in solid form and as circulatingcells, such as leukaemias, lymphomas and myeloproliferative diseases,for example acute myeloid, acute lymphoblastic, chronic lymphocytic,chronic myelogenous and hairy cell leukaemia, and AIDS-correlatedlymphomas, Hodgkin's lymphomas, non-Hodgkin's lymphomas, cutaneous Tcell lymphomas, Burkitt's lymphomas and lymphomas in the central nervoussystem.

These well-characterized diseases in humans can also occur with acomparable aetiology in other mammals and can likewise be treated therewith the compounds of the present invention.

The antibody-drug conjugates (ADCs) described herein and directedagainst CD123 can be used for the treatment of CD123-expressingdisorders, such as CD123-expressing cancer diseases. Typically, suchcancer cells exhibit measurable amounts of CD123 measured at the protein(e.g. using an immunoassay) or RNA level. Some of these cancer tissuesshow an elevated level of CD123 compared to non-cancerogenous tissue ofthe same type, preferably measured in the same patient. Optionally, theCD123 content is measured prior to the start of the cancer treatmentwith an antibody-drug conjugate (ADC) according to the invention(patient stratification). The antibody-drug conjugates (ADCs) directedagainst CD123 can be used for the treatment of CD123-expressingdisorders, such as CD123-expressing cancer diseases, such as tumours ofthe haematopoietic and lymphatic tissue or haematopoietic and lymphaticmalignant tumours. Examples of cancer diseases associated with CD123expression include myeloid diseases such as acute myeloid leukaemia(AML) and myelodysplastic syndrome (MDS). Other types of cancer includeB-cell acute lymphoblastic leukaemia (B-ALL), hairy cell leukaemia,blastic plasmacytoid dendritic cell neoplasm (BPDCN), Hodgkin'slymphoma, immature T-cell acute lymphoblastic leukaemia (immatureT-ALL), Burkitt's lymphoma, follicular lymphoma, chronic lymphocyticleukaemia (CLL), mantle cell lymphoma (MCL). Methods of the describedinvention comprise the treatment of patients suffering fromCD123-expressing cancer, the method comprising the administration of anantibody-drug conjugate (ADC) according to the invention.

The treatment of the cancer diseases mentioned above with the compoundsaccording to the invention comprises both a treatment of the solidtumours and a treatment of metastasizing or circulating forms thereof.

In the context of this invention, the term “treatment” or “treat” isused in the conventional sense and means attending to, caring for andnursing a patient with the aim of combating, reducing, attenuating oralleviating a disease or health abnormality, and improving the livingconditions impaired by this disease, as, for example, in the event of acancer.

The present invention thus further provides for the use of the compoundsof the invention for treatment and/or prevention of disorders,especially of the aforementioned disorders.

The present invention further provides for the use of the compounds ofthe invention for production of a medicament for treatment and/orprevention of disorders, especially of the aforementioned disorders.

The present invention further provides for the use of the compounds ofthe invention in a method for treatment and/or prevention of disorders,especially of the aforementioned disorders.

The present invention further provides a method of treatment and/orprevention of disorders, especially of the aforementioned disorders,using an effective amount of at least one of the compounds of theinvention.

The compounds of the invention can be used alone or, if required, incombination with one or more other pharmacologically active substances,provided that this combination does not lead to undesirable andunacceptable side effects. The present invention therefore furtherprovides medicaments comprising at least one of the compounds of theinvention and one or more further drugs, especially for treatment and/orprevention of the aforementioned disorders.

For example, the compounds of the present invention can be combined withknown anti-hyper-proliferative, cytostatic, cytotoxic orimmunotherapeutic substances for the treatment of cancer diseases.Examples of suitable combination drugs include:

131I-chTNT, abarelix, abiraterone, aclarubicin, adalimumab,ado-trastuzumab emtansine, afatinib, aflibercept, aldesleukin,alemtuzumab, alendronic acid, alitretinoin, altretamine, amifostine,aminoglutethimide, hexyl-5-aminolevulinate, amrubicin, amsacrine,anastrozole, ancestim, anethole dithiolethione, anetumab ravtansine,angiotensin II, antithrombin III, aprepitant, arcitumomab, arglabin,arsenic trioxide, asparaginase, atezolizumab, avelumab, axitinib,azacitidine, belotecan, bendamustine, besilesomab, belinostat,bevacizumab, bexarotene, bicalutamide, bisantrene, bleomycin,blinatumomab, bortezomib, buserelin, bosutinib, brentuximab vedotin,busulfan, cabazitaxel, cabozantinib, calcitonin, calcium folinate,calcium levofolinate, capecitabine, capromab, carbomazepine,carboplatin, carboquon, carfilzomib, carmofur, carmustine, catumaxomab,celecoxib, celmoleukin, ceritinib, cetuximab, chlorambucil,chlormadinone, chlormethine, cidofovir, cinacalcet, cisplatin,cladribine, clodronic acid, clofarabine, cobimetinib, copanlisib,crisantaspase, crizotinib, cyclophosphamide, cyproterone, cytarabine,dacarbazine, dactinomycin, daratumumab, dabrafenib, darolutamide,dasatinib, daunorubicin, decitabine, degarelix, denileukin-diftitox,denosumab, depreotide, deslorelin, dexrazoxane, dibrospidium chloride,dianhydrogalactitol, diclofenac, docetaxel, dolasetron, doxifluridine,doxorubicin, doxorubicin+estrone, dronabinol, durvalumab, edrecolomab,elliptinium acetate, endostatin, enocitabine, enzalutamide, epacadostat,epirubicin, epitiostanol, epoetin-alfa, epoetin-beta, epoetin-zeta,eptaplatin, eribulin, erlotinib, esomeprazole, estramustine, etoposide,ethylnyl oestradiol, everolimus, exemestane, fadrozole, fentanyl,fluoxymesterone, floxuridine, fludarabine, fluorouracil, flutamide,folic acid, formestane, fosaprepitant, fotemustine, fulvestrant,gadobutrol, gadoteridol, gadoteric acid meglumine salt, gadoversetamide,gadoxetic acid disodium salt (gd-EOB-DTPA disodium salt), galliumnitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab, glucarpidase,glutoxim, goserelin, granisetron, granulocyte colony stimulating factor(G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF),histamine dihydrochloride, histrelin, hydroxycarbamide, I-125 seeds,ibandronic acid, ibritumomab-tiuxetan, ibrutinib, idarubicin,ifosfamide, imatinib, imiquimod, improsulfan, indisetron, incadronicacid, ingenol mebutate, interferon-alfa, interferon-beta,interferon-gamma, iobitridol, iobenguane (123I), iomeprol, ipilimumab,irinotecan, itraconazole, ixabepilone, ixazomib, lanreotide,lansoprazole, lapatinib, lasocholine, lenalidomide, lenvatinib,lenograstim, lentinan, letrozole, leuprorelin, levamisole,levonorgestrel, levothyroxin-sodium, lipegfilgrastim, lisuride,lobaplatin, lomustine, lonidamine, masoprocol, medroxyprogesteron,megestrol, melarsoprol, melphalan, mepitiostan, mercaptopurine, mesna,methadone, methotrexate, methoxsalen, methylaminolevulinate,methylprednisolone, methyltestosterone, metirosine, mifamurtide,miltefosine, miriplatin, mitobronitol, mitoguazone, mitolactol,mitomycin, mitotan, mitoxantrone, mogamulizumab, molgramostim,mopidamol, morphine hydrochloride, morphine sulfate, nabilone,nabiximols, nafarelin, naloxone +pentazocine, naltrexone, nartograstim,necitumumab, nedaplatin, nelarabine, neridronic acid,netupitant/palonosetrone, nivolumab, nivolumab, pentetreotide,nilotinib, nilutamide, nimorazole, nimotuzumab, nimustine, nintedanib,nitracrine, nivolumab, obinutuzumab, octreotide, ofatumumab, olaparib,olaratumab, omacetaxin mepesuccinate, omeprazole, ondansetron, orgotein,orilotimod, osimertinib, oxaliplatin, oxycodone, oxymetholone,ozogamicin, p53 gene therapy, paclitaxel, palbociclib, palifermine,palladium-103 seed, palonosetron, pamidronic acid, panitumumab,panobinostat, pantoprazole, pazopanib, pegaspargase, pembrolizumab,Peg-interferon alfa-2b, pembrolizumab, pemetrexed, pentostatin,peplomycin, perflubutane, perfosfamide, pertuzumab, picibanil,pilocarpine, pirarubicin, pixantron, plerixafor, plicamycin, poliglusam,polyoestradiol phosphate, polyvinylpyrrolidone+sodium hyaluronate,polysaccharide-K, pomalidomide, ponatinib, porfimer-sodium,pralatrexate, prednimustine, prednisone, procarbazine, procodazole,propranolol, quinagolide, rabeprazole, racotumomab, radium-223 chloride,radotinib, raloxifene, raltitrexed, ramosetron, ramucirumab,ranimustine, rasburicase, razoxan, refametinib, regorafenib, risedronicacid, rhenium-186 etidronate, rituximab, rogaratinib, rolapitant,romidepsin, romurtid, roniciclib, samarium (153Sm) lexidronam,satumomab, secretin, siltuximab, sipuleucel-T, sizofiran, sobuzoxane,sodium glycididazole, sonidegib, sorafenib, stanozolol, streptozocin,sunitinib, talaporfin, talimogen laherparepvec, tamibarotene, tamoxifen,tapentadol, tasonermin, teceleukin, technetium (99mTc) nofetumomabmerpentan, 99mTc-HYNIC-[Tyr3]-octreotide, tegafur,tegafur+gimeracil+oteracil, temoporfin, temozolomide, temsirolimus,teniposide, testosterone, tetrofosmin, thalidomide, thiotepa,thymalfasin, thyrotropin alfa, tioguanine, tocilizumab, topotecan,toremifene, tositumomab, trabectedin, trametinib, tramadol, trastuzumab,treosulfan, tretinoin, trifluridine+tipiracil, trametinib, trilostane,triptorelin, trofosfamide, thrombopoietin, ubenimex, valrubicin,vandetanib, vapreotide, vatalanib, vemurafenib, vinblastine,vincristine, vindesine, vinflunine, vinorelbine, vismodegib, vorinostat,yttrium-90 glass microbeads, zinostatin, zinostatin stimalamer,zoledronic acid, zorubicin

In addition, the compounds of the present invention can be combined, forexample, with binders (e.g. antibodies) which, by way of example, canbind to the following targets: OX-40, CD137/4-1BB, DR3, IDO1/ID02,LAG-3, CD40.

Since a non-cell-permeable toxophore metabolite of an antibody-drugconjugate (ADC) should have no damaging effect on the cells of theadaptive immune system, the invention furthermore provides thecombination of an antibody-drug conjugate (ADC) according to theinvention with a cancer immunotherapy for use in the treatment of canceror tumours. The intrinsic mechanism of action of cytotoxic antibody-drugconjugates comprises the direct triggering of cell death of the tumourcells and thus the release of tumour antigens which may stimulate animmune response. Furthermore, there are indications that the KSPinhibitor toxophore class induces markers of immunogenic cell death(ICD) in vitro. Thus, the combination of the binder-drug conjugates(ADCs) of the present invention with one or more therapeutic approachesof cancer immunotherapy or with one or more active compounds, preferablyantibodies, directed against a molecular target of cancer immunotherapyrepresents a preferred method for treating cancer or tumours. i)Examples of therapeutic approaches of cancer immunotherapy compriseimmunomodulatory monoclonal antibodies and low-molecular weightsubstances directed against targets of cancer immunotherapy, vaccines,CAR T cells, bispecific T-cell-recruiting antibodies, oncolyticalviruses, cell-based vaccination approaches. ii) Examples of selectedtargets of cancer immunotherapy suitable for immunemodulatory monoclonalantibodies comprise CTLA-4, PD-1/PDL-1, OX-40, CD137, DR3, IDO1,IDO2,TDO2, LAG-3, TIM-3 CD40.,ICOS/ICOSL,TIGIT; GITR/GITRL, VISTA, CD70,CD27, HVEM/BTLA, CEACAM1, CEACAM6, ILDR2, CD73, CD47, B7H3, TLRs.Accordingly, combination of an antibody-drug conjugate (ADC) accordingto the invention with cancer immunotherapy could, on the one hand,render tumours with weak immunogenic properties more immunogenic andenhance the effectiveness of cancer immunotherapy, and furthermoreunfold long-lasting therapeutic action.

In addition, the compounds according to the invention can also be usedin combination with radiotherapy and/or surgical intervention.

Generally, the following aims can be pursued with the combination ofcompounds of the present invention with other cytostatically,cytotoxically or immunotherapeutically active agents:

-   -   improved efficacy in slowing the growth of a tumour, in reducing        its size or even in completely eliminating it, compared with        treatment with an individual active ingredient;    -   the possibility of using the chemotherapeutics used in a lower        dosage than in the case of monotherapy;    -   the possibility of a more tolerable therapy with fewer side        effects compared with individual administration;    -   the possibility of treatment of a broader spectrum of neoplastic        disorders;    -   the achievement of a higher rate of response to the therapy;    -   a longer survival time of the patient compared with present-day        standard therapy.

In addition, the compounds according to the invention can also be usedin combination with radiotherapy and/or surgical intervention.

The present invention further provides medicaments which comprise atleast one compound of the invention, typically together with one or moreinert, non-toxic, pharmaceutically suitable excipients, and for the usethereof for the aforementioned purposes.

The compounds of the invention can act systemically and/or locally. Forthis purpose, they can be administered in a suitable manner, for exampleparenterally, possibly inhalatively or as implants or stents.

The compounds of the invention can be administered in administrationforms suitable for these administration routes.

Parenteral administration can bypass an absorption step (for exampleintravenously, intraarterially, intracardially, intraspinally orintralumbally) or include an absorption (for example intramuscularly,subcutaneously, intracutaneously, percutaneously or intraperitoneally).Administration forms suitable for parenteral administration includepreparations for injection and infusion in the form of solutions,suspensions, emulsions or lyophilizates. Preference is given toparenteral administration, especially intravenous administration.

In general, it has been found to be advantageous in the case ofparenteral administration to administer amounts of about 0.1 to 20mg/kg, preferably about 0.3 to 7 mg/kg, of body weight to achieveeffective results.

It may nevertheless be necessary in some cases to deviate from thestated amounts, and specifically as a function of body weight, route ofadministration, individual response to the active ingredient, nature ofthe preparation and time at which or interval over which administrationtakes place. Thus in some cases it may be sufficient to manage with lessthan the abovementioned minimum amount, while in other cases the upperlimit mentioned must be exceeded. In the case of administration ofgreater amounts, it may be advisable to divide them into severalindividual doses over the day.

EXAMPLES

The examples which follow illustrate the invention. The invention is notrestricted to the examples.

Unless stated otherwise, the percentages in the tests and examples whichfollow are percentages by weight; parts are parts by weight. Solventratios, dilution ratios and concentration data for the liquid/liquidsolutions are based in each case on volume.

If no information with respect to a temperature at which the reaction iscarried out is given in the experimental descriptions, room temperatureis to be assumed.

Synthesis Routes:

By way of example for the working examples, the following schemes showillustrative synthesis routes leading to the working examples: Both thesynthesis sequence and the protective group strategy can be varied onthe route to the target compounds.

[a): for example sodium triacetoxy borohydride, acetic acid, DCM, RT; b)for example acetoxyacetyl chloride, NEt3, DCM, RT; c) for example LiOH,THF/water, RT; d) for example H₂, Pd-C, EtOH, RT; e) for exampleTeoc-OSu, NEt3, dioxane, RT; f) for example Fmoc-Cl,diisopropylethylamine, dioxane/water 2:1, RT]

[a): HATU, DMF, N,N-diisopropylethylamine, RT; b): H₂, 10% Pd-C,methanol, RT c):1,1′-[(1,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione,N,N-diisopropylethyl-amine, DMF, stirring at RT; d)2,2,2-trifluoroethanol, 4-8 equivalents of zinc chloride, 2-6 h at 50°C.; e) AK dissolved in PBS, under argon addition of 3-4 equivalents ofTCEP in PBS buffer and about 30 min stirring at RT, then addition of5-10 equivalents of compound D dissolved in DMSO, about 90 min ofstirring at RT, then rebuffering to pH 8 by means of PD 10 columnsequilibrated with PBS buffer (pH 8) (Sephadex® G-25, GE Healthcare),then stirring at RT overnight, then optionally purification by means ofPD 10 columns equilibrated with PBS buffer (pH 7.2) (Sephadex® G-25, GEHealthcare) and subsequent concentration by means of ultracentrifugationand setting of the concentration desired with PBS buffer (pH 7.2)]. Inthe case of in vivo batches, this is optionally followed by sterilefiltration.

A. Examples

Abbreviations and Acronyms:

-   -   ABCB1 ATP-binding cassette sub-family B member 1 (synonym for        P-gp and MDR1)    -   abs. absolute    -   Ac acetyl    -   ACN acetonitrile    -   aq. aqueous, aqueous solution    -   ATP adenosine triphosphate    -   BCRP breast cancer resistance protein, an efflux transporter    -   BEP 2-bromo-1-ethylpyridinium tetrafluoroborate    -   Boc tent-butoxycarbonyl    -   br. broad (in NMR)    -   Ex. Example    -   C concentration    -   ca. circa, about    -   CI chemical ionization (in MS)    -   d doublet (in NMR)    -   d day(s)    -   DAR drug-to-antibody ratio    -   TLC thin layer chromatography    -   DCI direct chemical ionization (in MS)    -   DCM dichloromethane    -   dd doublet of doublets (in NMR)    -   DMAP 4-N,N-dimethylaminopyridine    -   DME 1,2-dimethoxyethane    -   DMEM Dulbecco's Modified Eagle Medium (standardized nutrient        medium for cell culture)    -   DMF N,N-dimethylformamide    -   DMSO dimethyl sulfoxide    -   D/P dye (fluorescent dye)/protein ratio    -   DPBS, D-PBS, Dulbecco's phosphate-buffered salt solution    -   DSMZ Deutsche Sammlung von Mikroorganismen and Zellkulturen        (German Collection of Microorganisms and Cell Cultures)    -   PBS PBS =DPBS =D-PBS, pH 7.4, from Sigma, No D8537        -   Composition:        -   0.2 g KCl        -   0.2 g KH₂PO₄ (anhyd)        -   8.0 g NaCl        -   1.15 g Na₂HPO₄ (anhyd)        -   made up ad llwith H₂O    -   dt doublet of triplets (in NMR)    -   DTT DL-dithiothreitol    -   EDC N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride    -   EGFR epidermal growth factor receptor    -   El electron impact ionization (in MS)    -   ELISA enzyme-linked immunosorbent assay    -   eq. equivalent(s)    -   ESI electrospray ionization (in MS)    -   ESI-MicroTofq ESI- MicroTofq (name of the mass spectrometer with        Tof =time of flight and q =quadrupole)    -   FCS foetal calf serum    -   Fmoc (9H-fluoren-9-ylmethoxy)carbonyl    -   sat. saturated    -   GTP guanosine-5′-triphosphate    -   H hour(s)    -   HATU 0-(7-azabenzotriazol-1-yl)-N,N,N;N′-tetramethyluronium        hexafluorophosphate    -   HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid    -   HOAc acetic acid    -   HOAt 1-hydroxy-7-azabenzotriazole    -   HOBt 1-hydroxy-1H-benzotriazole hydrate    -   HOSu N-hydroxysuccinimide    -   HPLC high-pressure, high-performance liquid chromatography    -   IC₅₀ half-maximal inhibitory concentration    -   i.m. intramuscularly, administration into the muscle    -   i.v. intravenously, administration into the vein    -   KPL-4 human tumour cell line    -   conc. concentrated    -   LC-MS liquid chromatography-coupled mass spectrometry    -   LLC-PK1 cells Lewis lung carcinoma pork kidney cell line    -   L-MDR human MDR1 transfected LLC-PK1 cells    -   M multiplet (in NMR)    -   MDR1 Multidrug resistance protein 1    -   MeCN acetonitrile    -   Me methyl    -   min minute(s)    -   MOLM-13 human tumour cell line    -   MS mass spectrometry    -   MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium        bromide    -   MV-4-11 human tumour cell line    -   NCI-H292 human tumour cell line    -   NMM N-methylmorpholine    -   NMP N-methyl-2-pyrrolidinone    -   NMR nuclear magnetic resonance spectrometry    -   NMRI mouse strain originating from the Naval Medical Research        Institute (NMRI)    -   Nude mice experimental animals    -   NSCLC non small cell lung cancer    -   PBS phosphate-buffered salt solution    -   Pd/C palladium on activated charcoal    -   P-gp P-glycoprotein, a transporter protein    -   PNGaseF enzyme for cleaving sugar    -   quant. quantitative (in yield)    -   quart quartet (in NMR)    -   quint quintet (in NMR)    -   Rec-1 human tumour cell line    -   R_(f) retention index (in TLC)    -   RT room temperature    -   R_(t) retention time (in HPLC)    -   s singlet (in NMR)    -   s.c. subcutaneously, administration under the skin    -   SCID mice test mice with severe combined immunodeficiency    -   SK-HEP-1 human tumour cell line    -   t triplet (in NMR)    -   TBAF tetra-n-butylammonium fluoride    -   TEMPO (2,2,6,6-tetramethylpiperidin-1-yl)oxyl    -   Teoc trimethylsilylethoxycarbonyl    -   Teoc-OSu        1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione    -   tent tertiary    -   TFA trifluoroacetic acid    -   THF tetrahydrofuran    -   T3P® 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane        2,4,6-trioxide    -   UV ultraviolet spectrometry    -   v/v volume to volume ratio (of a solution)    -   Z benzyloxycarbonyl

HPLC and LC-MS Methods:

Method 1 (LC-MS):

Instrument: Waters ACQUITY SQD UPLC System; column: Waters Acquity UPLCHSS T3 1.8 μ 50×1 mm; mobile phase A: 1 l of water +0.25 ml of 99%strength formic acid; mobile phase B: 1 I of acetonitrile +0.25 ml of99% strength formic acid; gradient: 0.0 min 90% A→1.2 min 5% A→2.0 min5% A; oven: 50° C.; flow rate: 0.40 ml/min; UV detection: 208-400 nm.

Method 2 (LC-MS):

MS instrument type: Waters Synapt G2S; UPLC instrument type: WatersAcquity I-CLASS; column: Waters, BEH300, 2.1×150 mm, C18 1.7 μm; mobilephase A: 1 I of water +0.01% formic acid; mobile phase B: 1 I ofacetonitrile +0.01% formic acid; gradient: 0.0 min 2% B→1.5 min 2% B→8.5min 95% B→10.0 min 95% B; oven: 50° C.; flow rate: 0.50 ml/min; UVdetection: 220 nm

Method 3 (LC-MS):

MS instrument: Waters (Micromass) QM; HPLC instrument: Agilent 1100series; column: Agilent ZORBAX Extend-C18 3.0×50 mm 3.5 micron; mobilephase A: 1 I of water +0.01 mol of ammonium carbonate, mobile phase B: 1I of acetonitrile; gradient: 0.0 min 98% A→0.2 min 98% A→3.0 min 5%A→4.5 min 5% A ; oven: 40° C.; flow rate: 1.75 ml/min; UV detection: 210nm

Method 4 (LC-MS):

MS instrument type: Waters Synapt G2S; UPLC instrument type: WatersAcquity I-CLASS; column: Waters, HSST3, 2.1×50 mm, C18 1.8 pm; mobilephase A: 1 I of water +0.01% formic acid; mobile phase B: 1 I ofacetonitrile +0.01% formic acid; gradient: 0.0 min 10% B→0.3 min 10%B→1.7 min 95% B→2.5 min 95% B; oven: 50° C.; flow rate: 1.20 ml/min; UVdetection: 210 nm

Method 5 (LC-MS):

Instrument: Waters ACQUITY SQD UPLC System; column: Waters Acquity UPLCHSS T3 1.8 μ 50×1 mm; mobile phase A: 1 I of water +0.25 ml of 99%strength formic acid; mobile phase B: 1 I of acetonitrile +0.25 ml of99% strength formic acid; gradient: 0.0 min 95% A→6.0 min 5% A→7.5 min5% A; oven: 50° C.; flow rate: 0.35 ml/min; UV detection: 210-400 nm.

Method 6 (LC-MS):

Instrument: Micromass Quattro Premier with Waters UPLC Acquity; column:Thermo Hypersil GOLD 1.9 μ, 50×1 mm; mobile phase A: 1 I of water +0.5ml of 50% strength formic acid; mobile phase B: 1 I of acetonitrile +0.5ml of 50% strength formic acid; gradient: 0.0 min 97% A→0.5 min 97%A→3.2 min 5% A→4.0 min 5% A oven: 50° C.; flow rate: 0.3 ml/min; UVdetection: 210 nm.

Method 7 (LC-MS):

Instrument: Agilent MS Quad 6150; HPLC: Agilent 1290; column: WatersAcquity UPLC HSS T3 1.8 μ 50×2.1 mm; mobile phase A: 1 I of water +0.25ml of 99% strength formic acid; mobile phase B: 1 1 of acetonitrile+0.25 ml of 99% strength formic acid; gradient: 0.0 min 90% A→0.3 min90% A→1.7 min 5% A→3.0 min 5% A oven: 50° C.; flow rate: 1.20 ml/min; UVdetection: 205 — 305 nm.

Method 8 (LC-MS):

MS instrument type: Waters Synapt G2S; UPLC instrument type: WatersAcquity I-CLASS; column: Waters, HSST3, 2.1×50 mm, C18 1.8 pm; mobilephase A: 1 I of water +0.01% formic acid; mobile phase B: 1I ofacetonitrile +0.01% formic acid; gradient: 0.0 min 2% B→2.0 min 2%B→13.0 min 90% B→15.0 min 90% B; oven: 50° C.; flow rate: 1.20 ml/min;UV detection: 210 nm.

Method 9: (LC-MS prep. purification method)

MS instrument: Waters, HPLC instrument: Waters (column Waters X-BridgeC18, 19 mm×50 mm, 5 μm, eluent A: water +0.05% ammonia, mobile phase B:acetonitrile (ULC) with gradient; flow rate: 40 ml/min; UV detection:DAD; 210-400 nm).

or

MS instrument: Waters, HPLC instrument: Waters (column Phenomenex Luna 5μ C18(2) 100A, AXIA Tech. 50×21.2 mm, eluent A: water +0.05% formicacid, eluent B: acetonitrile (ULC) with gradient; flow rate: 40 ml/min;UV detection: DAD; 210-400 nm).

Method 10: (LC-MS analysis method)

MS instrument: Waters SQD; HPLC instrument: Waters UPLC; column: ZorbaxSB-Aq (Agilent), 50 mm×2.1 mm, 1.8 pm; mobile phase A: water +0,025%formic acid, eluent B:

acetonitrile (ULC) +0,025% formic acid; gradient: 0.0 min 98% A-0.9 min25% A-1.0 min 5% A-1.4 min 5% A-1.41 min 98% A-1.5 min 98% A; oven: 40°C.; flow rate: 0,600 ml/min; UV detection: DAD; 210 nm.

Method 11 (HPLC):

Instrument: HP1100 Series, column: Merck Chromolith SpeedROD RP-18e,50-4.6 mm, Cat. No.1.51450.0001, precolumn Chromolith Guard CartridgeKit, RP-18e, 5-4.6mm, Cat. No. 1.51470.0001; gradient: flow rate 5ml/min; injection volume 5 μl; solvent A: HClO4 (70%) in water (4 ml/l),solvent B: acetonitrile start 20% B, 0.50 min 20% B, 3.00 min 90% B,3.50 min 90% B, 3.51 min 20% B, 4.00 min 20% B, column temperature: 40°C., wavelength: 210 nm

Method 12 (LC-MS):

MS instrument type: Thermo Scientific FT-MS; instrument type UHPLC+:Thermo Scientific UltiMate 3000; column: Waters, HSST3, 2.1×75 mm, C181.8 pm; mobile phase A: 1 I of water +0.01% formic acid; mobile phase B:1 I of acetonitrile +0.01% formic acid; gradient: 0.0 min 10% B→2.5 min95% B→3.5 min 95% B; oven: 50° C.; flow rate: 0.90 ml/min; UV detection:210 nm/ optimum integration path 210-300 nm

Method 13: (LC-MS):

MS instrument: Waters (Micromass) Quattro Micro; instrument Waters UPLCAcquity; column: Waters BEH C18 1.7 μ 50×2.1 mm; mobile phase A: 1 I ofwater +0.01 mol of ammonium formate, mobile phase B: 1 I ofacetonitrile; gradient: 0.0 min 95% A→0.1 min 95% A→2.0 min 15% A→2.5min 15% A→2.51 min 10% A→3.0 min 10% A; oven: 40° C.; flow rate: 0.5ml/min; UV detection: 210 nm.

Method 14: (LC-MS):

MS instrument type: ThermoFisherScientific LTQ-Orbitrap-XL; HPLCinstrument type: Agilent 1200SL; column: Agilent, POROSHELL 120, 3×150mm, SB-C18 2.7 μm; mobile phase A: 1 I of water +0.1% trifluoroaceticacid; mobile phase B: 1 I of acetonitrile +0.1% trifluoroacetic acid;gradient: 0.0 min 2% B→0.3 min 2% B→5.0 min 95% B 10.0 min 95% B; oven:40° C.; flow rate: 0.75 ml/min; UV detection: 210 nm

All reactants or reagents whose preparation is not described explicitlyhereinafter were purchased commercially from generally accessiblesources. For all other reactants or reagents whose preparation likewiseis not described hereinafter and which were not commercially obtainableor were obtained from sources which are not generally accessible, areference is given to the published literature in which theirpreparation is described.

Starting Compounds and intermediates

Intermediate C52

(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrol-2-yl]-2,2-dimethylpropan-1-amine

10.00 g (49.01 mmol) of methyl 4-bromo-1H-pyrrole-2-carboxylate wereinitially charged in 100.0 ml of DMF, and 20.76 g (63.72 mmol) ofcaesium carbonate and 9.22 g (53.91 mmol) of benzyl bromide were added.The reaction mixture was stirred at RT overnight. The reaction mixturewas partitioned between water and ethyl acetate and the aqueous phasewas extracted with ethyl acetate. The combined organic phases were driedover magnesium sulfate and the solvent was evaporated under reducedpressure. The reaction was repreated with 90.0 g of methyl4-bromo-1H-pyrrole-2-carboxylate. The two combined reactions werepurified by preparative RP-HPLC (column: Daiso 300×100; 10p, flow rate:250 ml/min, MeCN/water). The solvents were evaporated under reducedpressure and the residue was dried under high vacuum. This gave 125.15 g(87% of theory) of the compound methyl1-benzyl-4-bromo-1H-pyrrole-2-carboxylate.

LC-MS (Method 1): R_(t)=1.18 min; MS (ESlpos): m/z=295 [M+H]⁺.

Under argon, 4.80 g (16.32 mmol) of methyl1-benzyl-4-bromo-1H-pyrrole-2-carboxylate were initially charged in DMF,and 3.61 g (22.85 mmol) of (2,5-difluorophenyl)boronic acid, 19.20 ml ofsaturated sodium carbonate solution and 1.33 g (1.63 mmol) of[1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II):dichloromethanewere added. The reaction mixture was stirred at 85° C. overnight. Thereaction mixture was filtered through Celite and the filter cake waswashed with ethyl acetate. The organic phase was extracted with waterand then washed with saturated NaCl solution. The organic phase wasdried over magnesium sulfate and the solvent was evaporated underreduced pressure. The residue was purified on silica gel (mobile phase:cyclohexane/ethyl acetate 100:3). The solvents were evaporated underreduced pressure and the residue was dried under high vacuum. This gave3.60 g (67% of theory) of the compound methyl1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carboxylate.

LC-MS (Method 7): R_(t)=1.59 min; MS (ESlpos): m/z=328 [M+H]⁺.

3.60 g (11.00 mmol) of methyl1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carboxylate were initiallycharged in 90.0 ml of THF, and 1.04 g (27.50 mmol) of lithium aluminiumhydride (2.4 M in THF) were added at 0° C. The reaction mixture wasstirred at 0° C. for 30 minutes. Saturated potassium sodium tartratesolution was added at 0° C. and the reaction mixture was admixed withethyl acetate. The organic phase was extracted three times with sat.potassium sodium tartrate solution. The organic phase was washed oncewith saturated NaCl solution and dried over magnesium sulfate. Thesolvent was evaporated under reduced pressure and the residue wasdissolved in 30.0 ml of dichloromethane. 3.38 g (32.99 mmol) ofmanganese(IV) oxide were added, and the mixture was stirred at RT for 48h. Another 2.20 g (21.47 mmol) of manganese(IV) oxide were added, andthe mixture was stirred at RT overnight. The reaction mixture wasfiltered through Celite and the filter cake was washed withdichloromethane. The solvent was evaporated under reduced pressure andthe residue 2.80 g of(1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carbaldehyde) was usedwithout further purification in the next step of the synthesis.

LC-MS (Method 7): R_(t)=1.48 min; MS (ESlpos): m/z=298 [M+H]⁺.

28.21 g (94.88 mmol) of1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carbaldehyde together with23.00 g (189.77 mmol) of (R)-2-methylpropane-2-sulfinamide wereinitially charged in 403.0 ml of absolute THF, and 67.42 g (237.21 mmol)of titanium(IV) isopropoxide were added and the mixture was stirred atRT overnight. 500 ml of saturated NaCl solution and 1000.0 ml of ethylacetate were added, and the mixture was stirred at RT for 1 h. Themixture was filtered through kieselguhr and the filtrate was washedtwice with saturated NaCl solution. The organic phase was dried overmagnesium sulfate, the solvent was evaporated under reduced pressure andthe residue was purified using Biotage Isolera (silica gel, column1500+340 g SNAP, flow rate 200 ml/min, ethyl acetate/cyclohexane 1:10).

LC-MS (Method 7): R_(t)=1.63 min; MS (ESlpos): m/z=401 [M+H]⁺.

25.00 g (62.42 mmol) of(R)-N-{(E/Z)-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-methylene}-2-methylpropane-2-sulfinamidewere initially charged in absolute THF under argon and cooled to −78° C.12.00 g (187.27 mmol) of tert-butyllithium (1.7 M solution in pentane)were then added at −78° C. and the mixture was stirred at thistemperature for 3 h. At −78° C., 71.4 ml of methanol and 214.3 ml ofsaturated ammonium chloride solution were then added successively andthe reaction mixture was allowed to warm to RT and stirred at RT for 1h. The mixture was diluted with ethyl acetate and washed with water. Theorganic phase was dried over magnesium sulfate and the solvent wasevaporated under reduced pressure. The residue(R)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-methylpropane-2-sulfinamidewas used without further purification in the next step of the synthesis.

LC-MS (Method 6): R_(t)=2.97 min; MS (ESlpos): m/z=459 [M+H]⁺.

28.00 g (61.05 mmol) of(R)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-methylpropane-2-sulfinamidewere initially charged in 186.7 ml of 1,4-dioxane, and 45.8 ml of HCl in1,4-dioxane solution (4.0 M) were then added. The reaction mixture wasstirred at RT for 2 h and the solvent was evaporated under reducedpressure. to The residue was purified by preparative HPLC (column:Kinetix 100×30; flow rate: 60 ml/min, MeCN/water). The acetonitrile wasevaporated under reduced pressure and dichloromethane was added to theaqueous residue. The organic phase was washed with sodium bicarbonatesolution and dried over magnesium sulfate. The solvent was evaporatedunder reduced pressure and the residue was dried under high vacuum. Thisgave 16.2 g (75% of theory) of the title compound.

LC-MS (Method 6): R_(t)=2.10 min; MS (ESlpos): m/z=338 [M-NH₂]+, 709[2M+H]⁺.

¹H-NMR (400 MHz, DMSO-d₆): δ [ppm]=0.87 (s, 9H), 1.53 (s, 2H), 3.59 (s,1H), 5.24 (d, 2H), 6.56 (s, 1H), 6.94 (m, 1H), 7.10 (d, 2H), 7.20 (m,1H), 7.26 (m, 2H), 7.34 (m, 2H), 7.46 (m, 1H).

Intermediate C58

(2S)-4-[{(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoicacid

4.3 g (12.2 mmol) of Intermediate C52 were dissolved in 525 ml of DCM,and 3.63 g (17.12 mmol) of sodium triacetoxyborohydride and 8.4 ml ofacetic acid were added. After 5 min of stirring at RT, 8.99 g (24.5mmol) of Intermediate L57 dissolved in 175 ml of DCM were added and thereaction was stirred at RT for a further 45 min. The reaction was thendiluted with 300 ml of DCM and washed twice with 100 ml of sodiumbicarbonate solution and once with saturated NaCl solution. The organicphase was dried over magnesium sulfate, the solvent was evaporated underreduced pressure and the residue was dried under high vacuum. Theresidue was then purified by preparative RP-HPLC (column: ChromatorexC18). After combination of the appropriate fractions, the solvent wasevaporated under reduced pressure and the residue was dried under highvacuum. This gave 4.6 g (61% of theory) of methyl(2S)-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoate.

LC-MS (Method 12): R_(t)=1.97 min; MS (ESlpos): m/z=614 (M+H)⁺.

2.06 g (3.36 mmol) of this intermediate were initially charged in 76 mlof DCM and acylated with 0.81 ml (7.17 mmol) of 2-chloro-2-oxoethylacetate in the presence of 2.1 ml of triethylamine. After 20 h ofstirring at RT, 0.36 ml of 2-chlor-2-oxoethyl acetate and 0.94 ml oftriethylamine were added and the reaction was stirred at RT for afurther 15 min. The mixture was then diluted with 500 ml of ethylacetate and extracted successively twice with 300 ml of 5% citric acid,twice with 300 ml of saturated sodium hydrogencarbonate solution andonce with 100 ml of saturated sodium chloride solution and then driedover magnesium sulfate and concentrated. Drying under high vacuum gave2.17 g (79% of theory) of the protected intermediate.

LC-MS (Method 1): R_(t)=1.48 min; MS (ESIpos): m/z=714 (M+H)⁺.

2.17 mg (2.64 mmol) of this intermediate were dissolved in 54 ml of THFand 27 ml of water, and 26 ml of a 2-molar lithium hydroxide solutionwere added. The mixture was stirred at RT for 30 min and then adjustedto a pH between 3 and 4 using 1.4 ml of TFA. The mixture wasconcentrated under reduced pressure. Once most of the THF had beendistilled off, the aqueous solution was extracted twice with DCM andthen concentrated to dryness under reduced pressure. The residue waspurified by preparative HPLC (column: Chromatorex C18). Aftercombination of the appropriate fractions, the solvent was evaporatedunder reduced pressure and the residue was lyophilized fromacetonitrile/water. This gave 1.1 g (63% of theory) of the titlecompound.

LC-MS (Method 1): R_(t)=1.34 min; MS (ESlpos): m/z=656 (M−H)⁻.

¹H-NMR (400 MHz, DMSO-d₆): δ [ppm]=0.03 (s, 9H), 0.58 (m, 1H), 0.74-0.92(m, 11H), 1.40 (m, 1H), 3.3 (m, 2H), 3.7 (m, 1H), 3.8-4.0 (m, 2H), 4.15(q, 2H), 4.9 and 5.2 (2d, 2H), 5.61 (s, 1H), 6.94 (m, 2H), 7.13-7.38 (m,7H), 7.48 (s, 1H), 7.60 (m, 1H), 12.35 (s, 1H).

Intermediate C66

2-(Trimethylsilyl)ethyl[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-{[2-(glycylamino)ethyl]amino}-1-oxobutan-2-yl]-carbamate

First, trifluoroacetic acid benzyl{2-[(2-aminoethyl)amino]-2-oxoethyl}carbamate was prepared fromN-[(benzyloxy)carbonyl]glycine and tert-butyl (2-aminoethyl)carbamate byconventional methods of peptide chemistry (HATU coupling and Boccleavage).

13 mg (0.036 mmol) of this intermediate and 25 mg (0.033 mmol) ofintermediate C58 were taken up in 3 mL of DMF, and 19 mg (0.05 mmol) ofHATU and 17 μl of N,N-diiso-propylethylamine were added. After 10 min ofstirring at RT, the mixture was concentrated and the residue waspurified by preparative HPLC. This gave 17.8 mg (60% of theory) of theintermediate.

LC-MS (Method 1): R_(t)=1.36 min; MS (ESlpos): m/z=891 (M+H)⁺.

17 mg (0.019 mmol) of this intermediate were dissolved in 10 ml ofethanol, palladium on carbon (10%) was added and the mixture washydrogenated at RT using hydrogen under standard pressure for 2 h. Thecatalyst was filtered off, the solvents were evaporated under reducedpressure and the residue was dried under high vacuum. This gave 9 mg(62% of theory) of the title compound.

LC-MS (Method 1): R_(t)=1.03 min; MS (ESlpos): m/z=757 (M+H)⁺.

Intermediate C118

tert-Butyl N-[(8S)-8-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]ethyl}-2,2-dimethyl-6,9-dioxo-5-oxa-7,10-diaza-2-siladodecan-12-yl]-D-alpha-glutaminate

The title compound was prepared by conventional methods of peptidechemistry by coupling intermediate L119 and intermediate C58 in thepresence of HATU and subsequent hydrogenolytical cleavage of the Zprotective group.

LC-MS (Method 1): R_(t)=1.05 min; MS (ESlpos): m/z=885 (M+H)⁺.

Intermediate C119

tert-Butylglycyl-N-[(8S)-8-{2-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]ethyl}-2,2-dimethyl-6,9-dioxo-5-oxa-7,10-diaza-2-siladodecan-12-yl]-D-alpha-glutaminate

Intermediate C119 was prepared by conventional methods of peptidechemistry by coupling 2,5-dioxopyrrolidin-1-ylN-[(benzyloxy)carbonyl]glycinate and intermediate C118 in the presenceof HATU and subsequent removal of the Z protective group byhydrogenation over 10% palladium on activated carbon inmethanol/dichloromethane at RT under hydrogen standard pressure.

LC-MS (Method 1): R_(t)=1.03 min; MS (ESlpos): m/z=942 (M+H)⁺.

Intermediate L1

Trifluoroacetic acidN-(2-aminoethyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide

The title compound was prepared by conventional methods of peptidechemistry from commercial (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)aceticacid and tert-butyl (2-aminoethyl) carbamate.

LC-MS (Method 1): R_(t)=0.17 min; MS (ESlpos): m/z=198 (M+H)⁺.

Intermediate L57

Methyl (25)-4-oxo-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoate

500.0 mg (2.72 mmol) of methyl L-asparaginate hydrochloride and 706.3 mg(2.72 mmol) of 2-(trimethylsilyl)ethyl2,5-dioxopyrrolidine-1-carboxylate were initially charged in 5.0 ml of1,4-dioxane, and 826.8 mg (8.17 mmol) of triethylamine were added. Thereaction mixture was stirred at RT overnight. The reaction mixture waspurified directly by preparative RP-HPLC (column: Reprosil 250×40; 10p,flow rate 50 ml/min, MeCN/water, 0.1% TFA). The solvents were thenevaporated under reduced pressure and the residue was dried under highvacuum. This gave 583.9 mg (74% of theory) of the compound(3S)-4-methoxy-4-oxo-3-({[2-(trimethylsilyhethoxy]carbonyl}amino)butanoicacid. LC-MS (Method 1): R_(t)=0.89 min; MS (ESlneg): m/z=290 (M−H)⁻.

592.9 mg of(35)-4-methoxy-4-oxo-3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoicacid were initially charged in 10.0 ml of 1,2-dimethoxyethane, themixture was cooled to −15° C. and 205.8 mg (2.04 mmol) of4-methylmorpholine and 277.9 mg (2.04 mmol) of isobutyl chloroformatewere added. The precipitate was filtered off with suction after 15 minand twice with in each case 10.0 ml of 1,2-dimethoxyethane. The filtratewas cooled to −10° C., and 115.5 mg (3.05 mmol) of sodium borohydridedissolved in 10 ml of water were added with vigorous stirring. Thephases were separated and the organic phase was washed once each withsaturated sodium hydrogencarbonate solution and saturated NaCl solution.The organic phase was dried over magnesium sulfate, the solvent wasevaporated under reduced pressure and the residue was dried under highvacuum. This gave 515.9 mg (91% of theory) of the compound methylN-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-homoserinate.

LC-MS (Method 1): R_(t)=0.87 min; MS (ESlpos): m/z=278 (M+H)⁺.

554.9 mg (2.00 mmol) of methylN-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-homoserinate were initiallycharged in 30.0 ml of dichloromethane, and 1.27 g (3.0 mmol) ofDess-Martin periodinane and 474.7 mg (6.00 mmol) of pyridine were added.The mixture was stirred at RT overnight. After 4 h, the reaction wasdiluted with dichloromethane and the organic phase was washed in eachcase three times with 10% strength Na₂S₂O₃ solution, 10% strength citricacid solution and saturated sodium hydrogencarbonate solution. Theorganic phase was dried over magnesium sulfate and the solvent wasevaporated under reduced pressure. This gave 565.7 mg (97% of theory) ofthe title compound.

¹H-NMR (400 MHz, DMSO-d₆): δ [ppm]=0.03 (s, 9H), 0.91 (m, 2H), 2.70-2.79(m, 1H), 2.88 (dd, 1H), 3.63 (s, 3H), 4.04 (m, 2H), 4.55 (m, 1H), 7.54(d, 1H), 9.60 (t, 1H).

Intermediate L119

Trifluoroacetic acid tert-butylN-(2-aminoethyl)-N²-[(benzyloxy)carbonyl]-D-alpha-glutaminate salt

Intermediate L119 was prepared by conventional methods of peptidechemistry by coupling commercial(2R)-2-{[(benzyloxy)carbonyl]amino}-5-tert-butoxy-5-oxopentanoic acid(1.00 g, 2.96 mmol) and tert-butyl (2-aminoethyl)carbamate (560 μl, 3.6mmol) in the presence of HATU and subsequent acidic removal of the Bocprotective group using 10% strength TFA in dichloromethane, withsubstantial preservation of the t-butyl ester protective group.Purification by preparative HPLC gave the title compound.

LC-MS (Method 1): R_(t)=0.62 min; MS (ESI-pos): m/z=380 (M+H)⁺.

Intermediate L120

Benzyl N-(2-aminoethyl)-N2-[(benzyloxy)carbonyl]-D-alpha-glutaminate

Intermediate L120 was prepared by conventional methods of peptidechemistry by coupling commercial(2R)-5-(benzyloxy)-2-{[(benzyloxy)carbonyl]amino}-5-oxopentanoic acid(830 mg, 2.23 mmol) and tert-butyl (2-aminoethyl)carbamate (420 μl, 2.7mmol) in the presence of HATU and subsequent acidic removal of the Bocprotective group using TFA in dichloromethane.

Intermediate F104

Trifluoroacetic acid(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-N-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)butanamidesalt

300 mg (0.456 mmol) of intermediate C58 were dissolved in 38 ml of DMF,and 142 mg (0.456 mmol) of intermediate L1 and 260 mg (0.684 mmol) ofHATU and 318 μl of N,N-diisopropylethylamine were added. The mixture wasstirred at RT for 60 min and then concentrated. The residue was purifiedby preparative HPLC, giving, after lyophilization, 338 mg (87% oftheory) of the protected intermediate.

LC-MS (Method 1): R_(t)=1.30 min; MS (ESIpos): m/z=837 (M+H)⁺.

In the second step, 338 mg (0.404 mmol) of this intermediate weredissolved in 40 ml of 2,2,2-trifluoroethanol. 330.2 mg (2.42 mmol) ofzinc chloride were added and the mixture was stirred at 50° C. for 3 h.708 mg (2.42 mmol) of ethylenediamine-N,N,N′,N′-tetraacetic acid and 4ml of a 0.1% strength aqueous trifluoroacetic acid solution were thenadded. The mixture was purified by preparative HPLC. Concentration ofthe appropriate fractions and lyophilization of the residue fromacetonitrile/water gave 265 mg (81% of theory) of to the title compound.

LC-MS (Method 1): R_(t)=0.82 min; MS (ESlpos): m/z=693 (M+H)⁺.

Intermediate F325

N-[2-({(2S)-2-Amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyhamino]butanoyl}amino)ethyl]-N²-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-D-alpha-glutaminetrifluoroacetic acid salt

30 mg (0.046 mmol) of intermediate C58 were coupled with 29 mg (0.055mmol) of trifluoroacetic acid benzylN-(2-aminoethyl)-N2-[(benzyloxy)carbonyl]-D-alpha-glutaminate salt(intermediate L120) in the presence of 1.5 equiv. of HATU and 3 equiv.of N,N-diisopropylethylamine. Purification by preparative HPLC gave 39.5mg (82% of theory) of the protected intermediate. From thisintermediate, initially the benzyl ester groups were removedhydrogenolytically. Subsequently, coupling with1-{2-[(2,5-dioxopyrrolidin-1-yl)-oxy]-2-oxoethyl}-1 H-pyrrole-2,5-dionewas carried out in DMF in the presence of 3 equiv. ofN,N-diisopropylethylamine. In the last step, 13.5 mg (0.012 mmol) ofthis intermediate were dissolved in 5 ml of 2,2,2-trifluoroethanol. 13mg (0.096 mmol) of zinc chloride were added and the mixture was stirredat 50° C. for 3 h. Subsequently, 28 mg (0.096 mmol) ofethylenediamine-N,N,N′,N′-tetraacetic acid were added. The mixture waspurified by preparative HPLC. Concentration of the appropriate fractionsand lyophilization of the residue from acetonitrile/water gave 9 mg (81%of theory) of the title compound.

LC-MS (Method 12): R_(t)=1.44 min; MS (ESlpos): m/z=822 (M+H)⁺.

B: Preparation of Antibody-Drug Conjugates (ADC)

B-1. General Method for Generation of Anti-CD123 and Anti-CXCR5Antibodies and Chimeric and Humanized Variants of Anti-CD123 andAnti-CXCR5 Antibodies

The protein sequence (amino acid sequence) of the antibodies used, forexample the anti-CD123 antibodies TPP-8987, TPP-8988 and TPP-9476 andthe anti-CXCR5 antibodies TPP-9024, TPP-9574 and TPP-9580, wastransformed into a DNA sequence that encodes the respective protein by amethod known to those skilled in the art and inserted into an expressionvector suitable for transient mammalian cell culture (as described byTom et al., Chapter 12 in Methods Express: Expression Systems, edited byMichael R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007).

B-2. General Method for Expression of Antibodies in Mammalian Cells

The antibodies, for example the anti-CD123 antibodies TPP-8987, TPP-8988and TPP-9476 and the anti-CXCR5 antibodies TPP-9024, TPP-9574 andTPP-9580, were produced in transient mammalian cell cultures, asdescribed by Tom et al., Chapter 12 in Methods Express: ExpressionSystems, edited by Michael R. Dyson and Yves Durocher, Scion PublishingLtd, 2007.

B-3. General Method for Purification of Antibodies from CellSupernatants

The antibodies, for example the anti-CD123 antibodies TPP-8987, TPP-8988and TPP-9476 and the anti-CXCR5 antibodies TPP-9024, TPP-9574 andTPP-9580, were obtained from the cell culture supernatants. The cellsupernatants were clarified by centrifugation of cells. The cellsupernatant was then purified by affinity chromatography on a MabSelectSure (GE Healthcare) chromatography column. To this end, the column wasequilibrated in DPBS pH 7.4 (Sigma/Aldrich), the cell supernatant wasapplied and the column was washed with about 10 column volumes of DPBSpH 7.4+500 mM sodium chloride. The antibodies were eluted in 50 mMsodium acetate pH 3.5+500 mM sodium chloride and then purified furtherby gel filtration chromatography on a Superdex 200 column (GEHealthcare) in DPBS pH 7.4.

Commercially available antibodies were purified by standardchromatography methods (protein A chromatography, preparative gelfiltration chromatography (SEC—size exclusion chromatography)) from thecommercial products.

B-4. General Method for Coupling to Cysteine Side Chains

The following antibodies were used in the coupling reactions:

anti-CD123 AK TPP-8987

anti-CD123 AK TPP-8988

anti-CD123 AK TPP-9476

anti-CXCRS AK TPP-9024

anti-CXCRS AK TPP-9574

anti-CXCRS AK TPP-9580

Small-Scale Coupling:

Between 2 and 5 equivalents of tris(2-carboxyethyl)phosphinehydrochloride (TCEP), dissolved in PBS buffer, were added to a solutionof 2-5 mg of the appropriate antibody in

PBS buffer in the concentration range between 1 mg/ml and 20 mg/ml,preferably in the range of about 5 mg/ml to 15 mg/ml, and the mixturewas stirred at RT for 30 min to 1 h. Subsequently, depending on theintended loading, from 2 to 12 equivalents, preferably about 5-10equivalents of the maleimide precursor compound to be coupled were addedas a solution in DMSO. Here, the amount of DMSO should not exceed 10% ofthe total volume. The mixture was stirred at RT for 60-240 min andsubsequently diluted with PBS buffer, which had been adjusted to pH 8beforehand, to a volume of 2.5-7.5 ml and then passed through a PD 10column (Sephadex® G-25, GE Healthcare) equilibrated with PBS buffer pH8, and eluted with PBS buffer pH 8. The eluate was stirred at RT underargon overnight. Subsequently, the solution was concentrated byultracentrifugation and rediluted with PBS buffer (pH 7.2).

Medium-Scale Coupling:

Under argon, a solution of 2-5 equivalents, preferably 3 equivalents, ofTCEP in PBS buffer (c ˜0.2-0.8 mg/ml, preferably 0.5 mg/ml) was added to20-200 mg of the antibody in question in PBS buffer (c ˜5-15 mg/ml). Themixture was stirred at RT for 30 min, and then 2-12, preferably 5-10,equivalents of a maleimide precursor compound dissolved in DMSO wereadded. After stirring at RT for a further 1.5 h-2 h, the mixture wasdiluted with PBS buffer which had been adjusted to pH 8 beforehand.

This solution was then applied to PD 10 columns (Sephadex® G-25, GEHealthcare) which had been equilibrated with PBS buffer pH 8 and waseluted with PBS buffer pH 8. The eluate was diluted with PBS buffer pH 8to a concentration of 1-5 mg/ml. This solution was stirred at RT underargon overnight. If required, the solution was then rebuffered to pH7.2. The ADC solution was concentrated by ultracentrifugation, redilutedwith PBS buffer (pH 7.2) and then optionally concentrated again to aconcentration of about 10 mg/ml.

In the structural formulae shown, AK can have the meaning taken from thetables according to the working examples:

anti-CD123 AK TPP-8987 (partially reduced)- 5§¹

anti-CD123 AK TPP-8988 (partially reduced)- 5§¹

anti-CD123 AK TPP-9476 (partially reduced)- 5§¹

anti-CXCRS AK TPP-9024 (partially reduced)- 5§¹

anti-CXCRS AK TPP-9574 (partially reduced)- 5§¹

anti-CXCRS AK TPP-9580 (partially reduced)- 5§¹

wherein

-   -   §¹ represents the linkage to the succinimide group or to any        isomeric hydrolysed open-chain succinamides or the alkylene        radical resulting therefrom, and    -   S represents the sulfur atom of a cysteine residue of the        partially reduced antibody.

Further Purification and Characterization of the Conjugates according tothe Invention

After the reaction, in some instances the reaction mixture wasconcentrated, for example by ultrafiltration, and then desalted andpurified by chromatography, for example using a

Sephadex® G-25 column. Elution was carried out, for example, withphosphate-buffered saline (PBS). The solution was then sterile filteredand frozen. Alternatively, the conjugate can be lyophilized.

B-7. Determination of the Antibody, the Toxophore Loading and theProportion of Open Cysteine Adducts

For protein identification in addition to molecular weight determinationafter deglycosylation and/or denaturing, a tryptic digestion was carriedout which, after denaturing, reduction and derivatization, confirms theidentity of the protein via the tryptic peptides found.

toxophore loading (in the tables referred to as DAR, drug-to-antibodyratio) of the PBS buffer solutions obtained of the conjugates describedin the working examples was determined as follows:

Determination of toxophore loading of lysine-linked ADCs was carried outby mass spectrometry determination of the molecular weights of theindividual conjugate species. Here, the antibody conjugates were firstdeglycosylated with PNGaseF, and the sample was acidified and, afterHPLC separation/desalting, analysed by mass spectrometry usingESI-MicroTof_(Q) (Bruker Daltonik). All spectra over the signal in theTIC (Total Ion Chromatogram) were added and the molecular weight of thedifferent conjugate species was calculated based on MaxEntdeconvolution. The DAR (=drug/antibody ratio) was then calculated aftersignal integration of the different species. For this purpose, the sumtotal of the integration results for all species weighted by thetoxophore count was divided by the sum total of the simply weightedintegration results for all species.

The toxophore loading of cysteine-linked conjugates was determined byreversed-phase chromatography of the reduced and denatured ADCs.Guanidinium hydrochloride (GuHCl) (28.6 mg) and a solution ofDL-dithiothreitol (DTT) (500 mM, 3 μl) were added to the ADC solution (1mg/ml, 50 μl). The mixture was incubated at 55° C. for one hour andanalysed by HPLC.

HPLC analysis was carried out on an Agilent 1260 HPLC system withdetection at 220 nm. A Polymer Laboratories PLRP-S polymericreversed-phase column (catalogue number PL1912-3802) (2.1 ×150 mm, 8 μmparticle size, 1000 Å) was used at a flow rate of 1 ml/min with thefollowing gradient: 0 min, 25% B; 3 min, 25% B; 28 min, 50% B. Eluent Aconsisted of 0.05% trifluoroacetic acid (TFA) in water, eluent B of0.05% trifluoroacetic acid in acetonitrile.

The detected peaks were assigned by retention time comparison with thelight chain (LO) and the heavy chain (HO) of the non-conjugatedantibody. Peaks detected exclusively in the conjugated sample wereassigned to the light chain with one toxophore (L1) and the heavy chainswith one, two and three toxophores (H1, H2, H3).

Average loading of the antibody with toxophores (referred to as DAR,drug-to-antibody ratio) was calculated from the peak areas determined byintegration as double the sum of HC load and LC load, where LC load iscalculated from the sum of the toxophore number-average weighedintegration results of all LC peaks divided by the sum of the singlyweighed integration results of all LC peaks, and where the HC load iscalculated from the sum of the toxophore number-average weighedintegration results of all HC peaks divided by the sum of the singlyweighed integration results of all HC peaks. In individual cases, it wasbe possible that, owing to co-elution of some peaks, it was not possibleto determine toxophore loading accurately.

In the cases where light and heavy chains could not be separatedsufficiently by HPLC, determination of toxophore loading ofcysteine-linked conjugates was carried out by mass spectrometrydetermination of the molecular weights of the individual conjugatespecies at light and heavy chain.

For this purpose, guanidinium hydrochloride (GuHCl) (28.6 mg) and asolution of DL-dithiothreitol (DTT) (500 mM, 3 μl) were added to the ADCsolution (1 mg/ml, 50 μl). The mixture was incubated for one hour at 55°C. and analysed by mass spectrometry after online desalting usingESI-MicroTofQ (Bruker Daltonik).

For the DAR determination, all spectra were added over the signal in theTIC (Total Ion Chromatogram), and the molecular weight of the differentconjugate species at light and heavy chain was calculated based onMaxEnt deconvolution. The average loading of the antibody withtoxophores was determined from the peak areas determined by integrationas twice the sum total of the HC loading and the LC loading. In thiscontext, the LC loading is calculated from the sum total of theintegration results for all LC peaks weighted by the toxophore count,divided by the sum total of the simply weighted integration results forall LC peaks, and the HC loading from the sum total of the integrationresults for all HC peaks weighted by the toxophore count, divided by thesum total of the simply weighted integration results for all HC peaks.

In the case of the open constructs, to determine the proportion of theopen cysteine adduct, the molecular weight area ratio of closed to opencysteine adduct (molecular weight delta 18 daltons) of all singlyconjugated light and heavy chain variants was determined. The mean ofall variants yielded the proportion of the open cysteine adduct.

B-8. Verification of the Antigen Binding of the ADC

The capability of the binder of binding to the target molecule waschecked after coupling had taken place. The person skilled in the art isfamiliar with various methods which can be used for this purpose; forexample, the affinity of the conjugate can be checked using ELISAtechnology or surface plasmon resonance analysis (BlAcore™ measurement).The conjugate concentration can be measured by the person skilled in theart using customary methods, for example for antibody conjugates byprotein determination. (see also Doronina et al.; Nature Biotechnol.2003; 21:778-784 and Poison et al., Blood 2007; 1102:616-623).

Working Examples ADCs

The ADCs shown in the structural formulae of the working examples andthe reference examples, which were coupled to the cysteine side chainsof the antibodies via maleimide radicals, are, depending on the linkerand the coupling procedure, mainly present in the ring-opened formshown. However, the preparation may comprise a small proportion of thering-closed form.

Examples 1

Exemplary Procedure A:

Under argon, a solution of 0.029 mg of TCEP in 0.05 ml of PBS buffer wasadded to 5 mg of the antibody in question in 0.5 ml of PBS (c=10 mg/ml).The mixture was stirred at RT for 30 min, and then 0.26 mg (0.00023mmol) of Intermediate F325 dissolved in 50 μl of DMSO was added. Afterstirring at RT for a further 90 min, the mixture was diluted to a volumeof 2.5 ml PBS buffer which had been adjusted to pH 8 beforehand and thenpassed through a PD 10 column (Sephadex® G-25, GE Healthcare)equilibrated with PBS buffer pH 8, and eluted with PBS buffer pH 8. Theeluate was then stirred at RT under argon overnight. This was followedby concentration by ultracentrifugation and redilution with PBS buffer(pH 7.2).

Exemplary Procedure B:

Under argon, a solution of 0.172 mg of TCEP in 0.3 ml of PBS buffer wasadded to 30 mg of the antibody in question in 3 ml of PBS (c=10 mg/ml).The mixture was stirred at RT for 30 min, and then 1.57 mg (0.0014 mmol)of Intermediate F325 dissolved in 300 μl of DMSO was added. Afterstirring at RT for a further 90 min, the mixture was diluted to a volumeof 5 ml PBS buffer which had been adjusted to pH 8 beforehand, thenpassed through a PD 10 column (Sephadex® G-25, GE Healthcare)equilibrated with PBS buffer pH 8, and eluted with PBS buffer pH 8. Theeluate was then stirred at RT under argon overnight. This solution wasthen applied to a PD 10 column (Sephadex® G-25, GE Healthcare) which hadbeen equilibrated with PBS buffer pH 7.2 and was eluted with PBS bufferpH 7.2. The eluate was then concentrated by ultracentrifugation,rediluted with PBS buffer (pH 7.2), reconcentrated and thensterile-filtered.

The following ADCs were prepared analogously to these procedures andcharacterized as indicated in the table:

Antibody C Example Target (TPP-) Procedure [mg/mL] DAR 1c-8987 CD1238987 B 7.63 3.7 1c-8988 CD123 8988 B 9.10 3.5 1c-9476 CD123 9476 B 8.53.1 1x-9024 CXCR5 9024 B 8.76 3.8 1x-9574 CXCR5 9574 B 9.48 4.1 1x-9580CXCR5 9580 B 9.25 4.2

Working Examples of Metabolites

Example M1N-(3-{[(2R)-2-Amino-2-carboxyethyl]sulfanyl}-3-carboxpropanoyhglycyl-N-[24{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}amino)ethyl]-D-alpha-glutaminetrifluoroacetic acid salt

Regioisomer 1, epimer mixture

Triethylamine (10 ml, 73 mmol) and then1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione (8.31g, 32.0 mmol) were added to a solution of methyl L-cysteinatehydrochloride (1:1) (5.00 g, 29.1 mmol) in 1,4-dioxane (200 ml). Thereaction was stirred at room temperature for 20 h. The solid was thenfiltered off and the filtrate was concentrated under high vacuum. Theresidue was purified by preparative HPLC.

210 μl (1.4 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene were added to asolution of the methylN-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteinate obtained (130 mg,465 μmol) and 3-bromo-4-methoxy-4-oxobutanoic acid (393 mg, 1.86 mmol)in DMF (6.5 ml), and the reaction was stirred at room temperature for 10min. The reaction was then concentrated under reduced pressure and theresidue was purified by preparative HPLC. The solvent was evaporatedunder reduced pressure and the residue was dried under high vacuum.

The resulting intermediate was coupled by conventional methods ofpeptide chemistry in the presence of HATU with intermediate C119. Themethyl esters were then hydrolysed by treating with a solution oflithium hydroxide in THF/water (1:1).

In the last step, 22 mg of the intermediate obtained were dissolved in10 ml of 2,2,2-trifluoroethanol. 34 mg (0.252 mmol) of zinc chloridewere added and the mixture was stirred at 50° C. for 1 h. 74 mg (0.252mmol) of ethylenediamine-N,N,N′,N′-tetraacetic acid, 10 ml of water and500 μl of TFA were then added. The mixture was filtered and the solventwas evaporated under reduced pressure. The residue was purified bypreparative HPLC. Concentration of the appropriate fractions andlyophilization of the residue from acetonitrile/water gave 13 mg (72% oftheory) of the title compound.

LC-MS (Method 5): Rt=2.44 min; MS (ESlneg): m/z=959 [M−H]⁻

Example M2N-(2-{[(2R)-2-Amino-2-carboxyethyl]sulfanyl}-3-carboxpropanoyhglycyl-N-[24{(25)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-(glycoloyhamino]butanoyl}amino)ethyl]-D-alpha-glutaminetrifluoroacetic acid salt

Regioisomer 2, epimer mixture

The title compounds M2 were prepared as epimer mixture analogously toExample Ml:

801 μl (5.4 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene were added to asolution of methyl N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteinate(1000 mg, 3.58 mmol) and 2-bromo-4-ethoxy-4-oxobutanoic acid (926 mg,4.11 mmol) in DMF (40 ml), and the reaction was stirred at roomtemperature for 2 h. The reaction was then concentrated under reducedpressure and the residue was purified by preparative HPLC.

The intermediate obtained was coupled by conventional methods of peptidechemistry in the presence of HATU and methylmorpholine with intermediateC119. The methyl ester and the ethyl ester were then hydrolysed bytreatment with a solution of lithium hydroxide in THF/water (1:1).

In the last step, 48 mg of this intermediate were dissolved in 5 ml of2,2,2-trifluoroethanol. 75 mg (0.550 mmol) of zinc chloride were addedand the mixture was stirred at 50° C. for 3 h. 160 mg (0.550 mmol) ofethylenediamine-N,N,N′,N′-tetraacetic acid, 2 ml of water and 20 μl ofTFA were then added. The solvent was concentrated under reduced pressureand the residue was purified by preparative HPLC. Concentration of theappropriate fractions and lyophilization of the residue fromacetonitrile/water gave 14 mg (39% of theory) of the title compound.

LC-MS (Method 5): Rt=2.41 min; MS (ESlneg): m/z=959 [M−H]⁻

For comparison, the reference-ADCs R1 were prepared. In section C, thesuperiority of the ADCs according to the invention from Example 1 overthe corresponding reference-ADCs R1 is shown in an exemplary manner.

Reference Examples R1

Exemplary Procedure

Under argon, a solution of 0.172 mg of TCEP in 300 μl of PBS buffer wasadded to 30 mg of the appropriate AK in 3 ml of PBS (c=10 mg/mL). Themixture was stirred at RT for 30 min, and 1.291 mg (1.6 μmol) ofintermediate F104, dissolved in 300 μl of DMSO, were then added. After afurther 90 min of stirring at RT, the mixture was diluted with 1.4 ml ofPBS buffer which had been adjusted to pH 8 beforehand.

This solution was then passed over PD 10 columns (Sephadex® G-25, GEHealthcare) equilibrated with PBS buffer pH 8 and eluted with PBS bufferpH 8. The eluate was diluted with PBS buffer pH 8 to a total volume of7.5 ml. This solution was stirred at RT under argon overnight and then,once more using PD-10 columns, rebuffered to pH 7.2. The eluate wasdiluted to a total volume of 14 ml. By ultracentrifugation, the mixturewas then concentrated to 2 ml, rediluted to 14 ml with PBS buffer (pH7.2) and re-concentrated to a volume of 3 ml. The sample is filteredthrough a centrifuge tube (Microsep Advance Centrifugal Device 0.2 pmSupor Membrane/from PALL). The ADC batch obtained was characterized asfollows:

The following ADCs were prepared analogously to these procedures andcharacterized as stated in the table:

Antibody C Example Target (TPP-) [mg/mL] DAR R1c-8987 CD123 8987 9.111.5 R1c-8988 CD123 8988 6.45 3.4 R1c-9476 CD123 9476 7.0 3.1 R1x-9024CXCR5 9024 9.47 3.7 R1x-9574 CXCR5 9574 7.57 3.9 R1x-9580 CXCR5 95809.10 3.7

Also for comparison, the metabolites Rm1 and Rm2 formed from theReference Examples R1 were prepared:

Reference Example Rm1 4-[(2-{[2-({(2S)-2-Amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}amino)ethyl]amino}-2-oxoethyl)amino]-2-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-4-oxobutanoicacid trifluoroacetic acid salt

Regioisomer 1 as epimer mixture:

First, methyl L-cysteinate hydrochloride (1:1) was converted into methylN-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteinate using1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione in DMFin the presence of N,N-diisopropylethylamine.

A little at a time, 208 μl (1.4 mmol) of1,8-diazabicyclo[5.4.0]undec-7-ene were added to a solution of methylN-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteinate (130 mg, 465 μmol)and 3-bromo-4-methoxy-4-oxobutanoic acid (393 mg, 1.86 mmol) in DMF (6.5ml), and the reaction was stirred at room temperature for 10 min. Themixture was then concentrated under reduced pressure and the residue waspurified by preparative HPLC. The solvent was evaporated under reducedpressure and the residue was dried under high vacuum.

The intermediate obtained was coupled with intermediate C66 byconventional methods of peptide chemistry in the presence of HATU. Themethyl esters were then hydrolysed by treatment with a solution oflithium hydroxide in THF/water (1:1).

In the last step, 18 mg of this intermediate were dissolved in 10.6 mlof 2,2,2-trifluoro-ethanol. 22 mg (0.16 mmol) of zinc chloride wereadded and the mixture was stirred at 50° C. for 2 h. 47 mg (0.16 mmol)of ethylenediamine-N,N,N′,N′-tetraacetic acid and 2 ml of water and 2-3drops of TFA were then added. The mixture was filtered and the solventwas evaporated under reduced pressure. The residue was purified bypreparative HPLC. Concentration of the appropriate fractions andlyophilization of the residue from acetonitrile/water gave 10.5 mg(78.5% of theory) of the title compound (isomer 2) as regioisomermixture.

LC-MS (Method 5): Rt=2.43 min; MS (ESI-pos): m/z=832 [M+H]+

Reference Example Rm2 4-[(2-{[2-({(25)-2-Amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}amino)ethyl]amino}-2-oxoethyl)amino]-3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-4-oxobutanoicacid trifluoroacetic acid salt

Isomer 2 as epimer mixture:

First, methyl L-cysteinate hydrochloride (1:1) was converted into methylN-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteinate using1-({[2-(trimethylsilyhethoxy]carbonyI}-oxy)pyrrolidine-2,5-dione in DMFin the presence of N,N-diisopropylethylamine.

801 μl (5.4 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene were added to asolution of methyl N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteinate(1000 mg, 3.58 mmol) and 2-bromo-4-ethoxy-4-oxobutanoic acid (926 mg,4.11 mmol) in DMF (40 ml), and the reaction was stirred at roomtemperature for 2 h. The mixture was then concentrated under reducedpressure and the residue was purified by preparative HPLC. The solventwas evaporated under reduced pressure and the residue was dried underhigh vacuum.

The intermediate obtained was coupled with intermediate C66 byconventional methods of peptide chemistry in the presence of HATU. Themethyl ester and the ethyl ester were then hydrolysed by treatment witha solution of lithium hydroxide in THF/water (1:1).

In the last step, 24 mg of this intermediate were dissolved in 6.4 ml of2,2,2-trifluoro-ethanol. 28.5 mg (0.21 mmol) of zinc chloride were addedand the mixture was stirred at 50° C. for 2 h. 61 mg (0.21 mmol) ofethylenediamine-N,N,N′,N′-tetraacetic acid and 2 ml of water and 2-3drops of TFA were then added. The mixture was filtered and the solventwas evaporated under reduced pressure. The residue was purified bypreparative HPLC. Concentration of the appropriate fractions andlyophilization of the residue from acetonitrile/water gave 14.5 mg (71%of theory) of the title compound.

LC-MS (Method 5): Rt=2.41 min; MS (ESI-pos): m/z=832 [M+H]+

C: Assessment of Biological Efficacy

The biological activity of the compounds according to the invention canbe shown in the assays described below:

C-1a: Determination of the Cytotoxic Effect of the ADCs Directed againstCD123 and CXCR5

The analysis of the cytotoxic effect of the exemplary ADCs was carriedout with various cell lines:

NCl-H292: human mucoepidermoid lung carcinoma cells, ATCC-CRL-1848,standard medium: RPMI 1640 (Biochrom; #FG1215, stab. glutamine) +10% FCS(Sigma; #F2442), TWEAKR-positive; EGFR-positive,

KPL4: human breast cancer cell line, Bayer Pharma AG (identity checkedand confirmed on 19.7.2012 at DSMZ), standard medium: RPMI 1640 (fromGibco; #21875-059, stab. L-glutamine) +10% heat inactivated FCS (Gibco,No. 10500-064); HER2-positive.

SK-HEP-1: human liver cell cancer line, ATCC No. HTB-52, standardmedium: MEM with Earle's salts +Glutamax I (Invitrogen 41090) +10% heatinactivated FCS (from Gibco, No. 10500-064); TWEAKR-positive

MOLM-13: human acute monocytic leukaemia cells (AML-M5a), DSMZ, No. ACC554, standard medium: RPMI 1640 (from Gibco; #21875-059, stab.L-glutamine) +20% heat inactivated FCS (Gibco, No. 10500-064);CD123-positive.

MV-4-11: human biphenotypic B myelomonocytic leukaemia cells obtainedfrom peripheral blood, ATCC-CRL-9591, standard medium: IMDM (ATCC:30-2005), +10% heat inactivated FCS (Gibco, No. 10500-064);CD123-positive

NB4: human acute promyelocytic leukaemia cells obtained from bonemarrow, DSMZ, No. ACC 207, standard medium: RPMI 1640 +GlutaMAX I(Invitrogen 61870) +10% heat inactivated FCS (Gibco, No. 10500-064) +2.5g of glucose (20% glucose solution, Gibco, No.19002) +10 mM Hepes(Invitrogen 15630) +1 mM sodium pyruvate (Invitrogen 11360);CD123-negative

Rec-1: human mantle cell lymphoma cells (B cell non-Hodgkin's lymphoma)ATCC CRL-3004, standard medium: RPMI 1640 +GlutaMAX I (Invitrogen 61870)+10% heat inactivated FCS (Gibco, No. 10500-064) CXCRS-positive

The cells were cultivated by the standard method as stated by theAmerican Tissue Culture Collection (ATCC) or the Leibniz-lnstitutDSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ)for the cell lines in question.

MTT Assay

The cells were cultivated by the standard method, with the growth mediaspecified in section C-1. The test was carried out by detaching thecells with a solution of Accutase in PBS (from Biochrom AG #L2143),pelletizing, resuspending in culture medium, counting and sowing into a96-well culture plate with white bottom (from Costar #3610) (NCI H292:2500 cells/well; SK-HEP-1: 1000 cells/well; KPL-4: 1200 cells/well; intotal volume 100 μl). The cells were then incubated in an incubator at37° C. and 5% carbon dioxide. After 48 h, the medium was replaced. Theantibody drug conjugates in 10 μl of culture medium in concentrationsfrom 10⁻⁵M to 10⁻¹³M were then pipetted to the cells (in triplicate),and the assay was then incubated in an incubator at 37° C. and 5% carbondioxide. The suspension cells were counted and sown into a 96-wellculture plate with white bottom (from Costar #3610) (#3610) (MOLM-13:2000 cells/well; NB4: 7000 cells/well; MV-4-11: 5000 cells/well in atotal volume of 100 μl). After 6 hours of incubation at 37° C. and 5%carbon dioxide, the medium was changed and the antibody-drug conjugatesor metabolites were added by pipette in 10 μl of culture medium inconcentrations of 10⁻⁵M to 10⁻¹³M to the cells (triplicates) in 90 μl.The batch was incubated in an incubator at 37° C. and 5% carbon dioxide.After 96 h, the cell proliferation was detected using the MTT assay(ATCC, Manassas, Va., USA, catalogue No. 30-1010K). To this end, the MTTreagent was incubated with the cells for 4 h, followed by lysis of thecells overnight by addition of the detergent. The dye formed wasdetected at 570 nm (Infinite M1000 pro, Tecan). The measured data wereused to calculate the IC₅₀ of the growth inhibition using the DRC (doseresponse curve). The proliferation of cells which were not treated withtest substance but were otherwise identically treated was defined as the100% figure.

Table 1a below sets out the IC₅₀ values for representative workingexamples from this assay:

TABLE 1a MV-4-11 MOLM-13 Rec-1 Example IC₅₀ [M] IC₅₀ [M] IC₅₀ [M]1c-8987 1.26E−09 2.10E−10 1c-8988 2.94E−08 3.02E−11 1c-9476 2.78E−092.85E−10 1x-9024 1.34E−10 1x-9574 2.94E−11 1x-9580 2.78E−09

Table 1 b below lists the IC₅₀ values for representative referenceexamples from this assay.

TABLE 1b MV-4-11 MOLM-13 Rec-1 Example IC₅₀ [M] IC₅₀ [M] IC₅₀ [M]R1c-8987 1.33E−07 7.57E−08 R1c-8988 3.27E−08 4.26E−09 R1c-9476 5.30E−093.04E−10 R1x-9024 1.84E−10 R1x-9574 2.62E−10 R1x-9580 7.16E−11

The activity data reported relate to the working examples described inthe present experimental section, with the drug/mAB ratios indicated.The values may possibly deviate for different drug/mAB ratios. The IC50values are means of several independent experiments or individualvalues. The action of the antibody drug conjugates was selective for therespective isotype control comprising the respective linker andtoxophore. In addition, the target specificity of the antibody-drugconjugates directed against CD123 was demonstrated by testing with aCD123-negative cell (NB4).

C-1 b: Determination of the Inhibition of the Kinesin Spindle ProteinKSP/ Eg5 by Selected Examples

The motor domain of the human kinesin spindle protein KSP/Eg5 (tebu-bio/Cytoskeleton Inc, No. 027EG01-XL) was incubated in a concentration of 10nM with microtubuli (bovine or porcine, tebu-bio/Cytoskeleton Inc)stabilized with 50 μg/ml taxol (Sigma No. T7191-5MG) for 5 min at RT in15 mM PIPES, pH 6.8 (5 mM MgCl₂ and 10 mM DTT, Sigma). The freshlyprepared mixture was aliquoted into a 384 MTP (from Corning). Theinhibitors to be examined at concentrations of 1.0×10-6 M to 1.0×10-13 Mand ATP (final concentration 500 μM, Sigma) were then added. Incubationwas at RT for 2 h. ATPase to activity was detected by detecting theinorganic phosphate formed using malachite green

(Biomol). After addition of the reagent, the assay was incubated at RTfor 50 min prior to detection of the absorption at a wavelength of 620nm. The positive controls used were monastrol (Sigma, M8515-1mg) andispinesib (AdooQ Bioscience A10486). The individual data of thedose-activity curve are eight-fold determinations. The IC₅₀ values aremeans of two independent experiments. The 100% control was the samplewhich had not been treated with inhibitors.

Table 2 below lists the IC₅₀ values of representative working examplesfrom the assay described and summarizes the corresponding cytotoxicitydata (MTT assay):

TABLE 2 KSP-Assay NCI-H292 SKHep-1 KPL-4 MV 4-11 MOLM-13 Example IC₅₀[M] IC₅₀ [M] IC₅₀ [M] IC₅₀ [M] IC₅₀ [M] IC₅₀ [M] Rm1 9.44E−10 6.36E−081.90E−08 M1 2.13E−09 5.00E−07 9.88E−08 5.00E−07 3.02E−07 1.25E−07 Rm22.03E−09 2.76E−07 8.90E−08 M2 4.67E−10 1.85E−07 2.15E−07

The activity data reported relate to the working examples described inthe present experimental section.

C-2a Internalization Assay

Internalization is a key process which enables specific and efficientprovision of the cytotoxic payload in antigen-expressing cancer cellsvia antibody drug conjugates (ADC). This process is monitored viafluorescent labelling of specific antibodies and an isotype controlantibody. First, the fluorescent dye was conjugated to lysines of theantibody.

Conjugation was carried out using a two-fold molar excess of CypHer 5Emono NHS ester (Batch 357392, GE Healthcare) at pH 8.3. After thecoupling, the reaction mixture was purified by gel chromatography (ZebaSpin Desalting Columns, 40K, Thermo Scientific, No. 87768; elutionbuffer: DULBECCO′S PBS, Sigma-Aldrich, No. D8537), to eliminate excessdye and to adjust the pH. The protein solution was concentrated usingVIVASPIN 500 columns (Sartorius stedim biotec). The dye load of theantibody was determined by means of spectrophotometry analysis (fromNanoDrop) and subsequent calculation (D/P =A_(dye) ε_(protein):(A₂₈₀-0.16A_(dye))ε_(dye)).

The dye load of the antibodies examined here and the isotype controlwere of a comparable order of magnitude. In cell binding assays, it wasconfirmed that the coupling did not lead to any change in the affinityof the antibodies.

The labelled antibodies were used for the internalization assay. Priorto the start of the treatment, cells (2×10⁴/well) were sown in 100 μlmedium in a 96-well MTP (fat, black, clear bottom No 4308776, fromApplied Biosystems). After 18 h of incubation at 37° C./5%/CO₂, themedium was replaced and labelled antibodies were added in differentconcentrations (10, 5, 2.5, 1, 0.1 μg/ml). The same treatment protocolwas applied to the labelled isotype control (negative control). Thechosen incubation times were 0 h, 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 6h and 24 h. The fluorescence measurement was carried out using theInCellAnalyzer 1000 (from GE Healthcare). This was followed by kineticevaluation via measurement of the parameters granule counts/cell andtotal granule intensity/cell.

Following binding to the receptor, the antibodies were examined fortheir internalization capacity. For this purpose, tumour cells withdifferent receptor expression levels were chosen. A target-mediatedhighly specific internalization was observed with the antibodies of theinvention, whereas the isotype control showed no internalization.

C-2b: Internalization Assay with Suspended Cells

Coupling of the fluorescent dye was carried out as described in sectionC-2. The antigen to be examined is expressed by haematopoieticsuspension cells; consequently, the internalization was examined in anFACS-based internalization assay. Cells having different targetexpression levels were examined. The cells (5×10⁴/well) were sown in a96-MTP (Greiner bio-one, CELLSTAR, 650 180, U-bottom) in a total volumeof 100 μl. After addition of the target-specific antibody in a finalconcentration of 10 μg/ml, the batches were incubated at 37° C. fordifferent periods of time (1h, 2h, 6h, in triplicate). The isotypecontrol was treated under identical conditions. A parallel batch wastreated and incubated constantly at 4° C. (negative control). FACSanalysis was carried out using the Guava flow cytometer (Millipore).Kinetic evaluation was carried out by measuring the fluorescenceintensity, and evaluation took place using the guavaSoft 2.6 software(Millipore). For the targets and target-specific antibodies describedhere, a significant and specific internalization was detected in variouscells; the isotype controls showed no internalization.

C-2c: Co-Localization: Assays of the Anti-CD123 Antibodies

Owing to the linker, the active metabolite of the antibody-drugconjugate is generated by lysosomal degradation. Accordingly,intracellular trafficking after internalization has taken place is ofessential importance. Studies about the co-localization of the antibodyusing labels specific for the lysosomal organelle (e.g. surfacemolecules or small GTPases) allow the selection of antibodies having thedesired profile. To this end, target-positive cells (5×10⁴/well) in atotal volume of 100 μl were sown into a 96-MTP (Greiner bio-one,CELLSTAR, 650 180, U-bottom). Following addition of theCypHer5E-labelled anti-target antibody (final concentation 20 μg/ml),the batches (duplicates per point in time) were incubated at 37° C. for30 min, 2 h and 6 h in an incubator (5% CO₂). 30 min prior to the end ofthe chosen incubation time, the lysosome-specific label was added to thebatches to be examined. The lysosomes were stained with CytoPainterLysoGreen indicator reagent (final concentration 1:2000; abcam,ab176826). After incubation, 200 μl of ice-cold FACS buffer (DULBECCO′SPBS, Sigma-Aldrich, No. D8537 +3% FBS heat inactivated FBS, Gibco, No.10500-064) were added and the cell suspension was centrifuged at 400×gand 4° C. for 5 min. The cell pellet was resuspended in 300 μl ice-coldFACS buffer and centrifuged again (4 min, 400×g at 4° C.). Aftercentrifugation, the supernatant was discarded and the cell pellet wastaken up in 30 μl of ice-cold FACS buffer. The samples were thenimmediately subjected to FACS/image analysis (FlowSight amnis,Millipore). Co-localization was evaluated using a special software(co-localization software IDEAS Application v6.1). Table 3 summarizesthe results from this assay for examples of the anti-CD123 antibodies.

TABLE 3 Example Co-localization [%] TPP-9476 29 TPP-8997 28 TPP-8988 417G3 10 Isotype control 0.2

The antibodies TPP-8987 and TPP-9476 exhibit a markedly improved profilecompared to the parental murine antibody.

C-3 In vitro Tests for Determining Cell Permeability

The cell permeability of a substance can be investigated by means of invitro testing in a flux assay using Caco-2 cells [M.D. Troutman and D.R.Thakker, Pharm. Res. 20 (8), 1210-1224 (2003)]. For this purpose, thecells were cultured for 15-16 days on 24-well filter plates. For thedetermination of permeation, the respective test substance was appliedin a HEPES buffer to the cells either apically (A) or basally (B) andincubated for 2 hours. After 0 hours and after 2 hours, samples weretaken from the cis and trans compartments. The samples were separated byHPLC (Agilent 1200, Boblingen, Germany) using reverse phase columns. TheHPLC system was coupled via a Turbo Ion Spray Interface to a TripleQuadropole mass spectrometer API 4000 (AB SCIEX Deutschland GmbH,Darmstadt, Germany). The permeability was evaluated on the basis of aP_(app) value, which was calculated using the formula published bySchwab et al. [D. Schwab et al., J. Med. Chem. 46, 1716-1725 (2003)]. Asubstance was classified as actively transported when the ratio ofP_(app) (B-A) to P_(app) (A-B) (efflux ratio) was >2 or <0.5.

Of critical importance for toxophores which are released intracellularlyis the permeability from B to A [P_(app) (B-A)] and the ratio of P_(app)(B-A) to P_(app) (A-B) (efflux ratio): The lower this permeability, theslower the active and passive transport processes of the substancethrough the monolayer of Caco-2 cells. If the efflux ratio, moreover,gives no indications as to active transport, the substance, followingintracellular release, can remain in the cell for longer. As aconsequence, the time available for interaction with the biochemicaltarget (here: kinesin spindle protein KSP/Eg5) also increases.

Table 4 below sets out permeability data for representative workingexamples from this assay:

TABLE 4 Working P_(app) (B-A) Efflux Example [nm/s] ratio M1 1.2 0.8 M21.1 1.6 Rm1 13.0 9.6 Rm2 13.2 11.9

The metabolites M1 and M2, which can be formed from the ADCs accordingto the invention in Example 1, exhibit both reduced transport from thecell and a reduced efflux ratio compared with the reference metabolitesRM1 and RM2, which are formed from the ADC in Reference Example 1.

C-4 In Vitro Tests for Determining the Substrate Properties forP-Glycoprotein (P-gp)

Many tumour cells express transporter proteins for drugs, and thisfrequently accompanies the development of resistance towardscytostatics. Substances which are not substrates of such transporterproteins, such as P-glycoprotein (P-gp) or BCRP, for example, couldtherefore exhibit an improved activity profile.

The substrate properties of a substance for P-gp (ABCB1) were determinedby means of a flux assay using LLC-PK1 cells which overexpress P-gp(L-MDR1 cells) [A.H. Schinkel et al., J. Clin. Invest. 96, 1698-1705(1995)]. For this purpose, the LLC-PK1 cells or L-MDR1 cells werecultured on 96-well filter plates for 3-4 days. For determination of thepermeation, the respective test substance, alone or in the presence ofan inhibitor (such as ivermectin or verapamil, for example), was appliedin a HEPES buffer to the cells either apically (A) or basally (B) andincubated for 2 hours. After 0 hours and after 2 hours, samples weretaken from the cis and trans compartments. The samples were separated byHPLC using reverse phase columns. The HPLC system was coupled via aTurbo Ion Spray Interface to an API 3000 triple quadropole massspectrometer (Applied Biosystems Applera, Darmstadt, Germany). Thepermeability was evaluated on the basis of a P_(app) value, which wascalculated using the formula published by Schwab et al. [D. Schwab etal., J. Med. Chem. 46, 1716-1725 (2003)]. A substance was classified asP-gp substrate when the efflux ratio of P_(app) (B-A) to P_(app) (A-B)was >2.

As further criteria for the evaluation of the P-gp substrate properties,the efflux ratios in L-MDR1 and LLC-PK1 cells or the efflux ratio in thepresence or absence of an inhibitor may be compared. If these valuesdiffer by a factor of more than 2, the substance in question is a P-gpsubstrate.

C-5a: Identification of the ADC Metabolites after Internalization InVitro

Description of the Method:

Internalization studies with immunoconjugates were carried out toanalyse metabolites formed intracellularly. To this end, human lungtumour cells NCI H292 (3×10⁵/well) were sown in 6-well plates andincubated overnight (37° C., 5% CO₂). The cells were treated with 10μg/ml (66 nM) of the ADC to be examined. Internalization was carried outat 37° C. and 5% CO₂. Cell samples were taken for further analysis atvarious times (0, 4, 24, 48, 72 h). First of all, the supernatants(about 5 ml) were harvested and, after centrifugation (2 min, RT, 1000rpm Heraeus Variofuge 3.0R), stored at −80° C. The cells were washedwith PBS and detached with Accutase, and the cell number was determined.After another washing, a defined number of cells (2×10⁵) was treatedwith 100 ml of lysis buffer (Mammalian Cell Lysis Kit (Sigma MCL1) andincubated with continuous shaking (Thermomixer, 15 min, 4° C., 650 rpm)in Protein LoBind tubes (Eppendorf Cat. No. 0030 108.116). After theincubation, the lysate was centrifuged (10 min, 4° C., 12000 g,eppendorf 5415R) and the supernatant was harvested. The supernatantobtained was stored at −80° C. All samples were then analysed asfollows.

The compounds in the culture supernatant or cell lysate were analysedafter precipitation of the proteins with methanol or acetonitrile byhigh-pressure liquid chromatography (HPLC) coupled to atriple-quadrupole mass spectrometer (MS).

For workup of 50 μl of culture supernatant/cell lysate, 150 μl ofprecipitation reagent (methanol) are added and the mixture is shaken for10 seconds. The precipitation reagent contains an internal standard(ISTD) in a suitable concentration (generally in the range of 20-100μg/I). After centrifugation at 1881 g for 10 minutes, the supernatant istransferred into an autosampler vial, made up with 300 μl of a buffermatched to the eluent and shaken again and centrifuged at 1881 g for 10min.

The cell lysate and supernatant samples are finally analysed using theHPLC-coupled AP14200 triple-quadrupole mass spectrometer from AB SCIEXDeutschland GmbH.

For calibration, blank lysate or blank supernatant is admixed withappropriate concentrations (0.1-1000 μg/I). The detection limit (LLOQ)is about 0.2 μg/I.

Quality controls for testing validity contain 4 and 40 μg/I.

C-5b: Identification of the ADC Metabolites In Vivo

Analysis for Quantification of the Potential Metabolites

After i.v. administration of 10 mg/kg of different conjugates accordingto the invention to xenograft mice, it is possible to measure theplasma, tumour, liver, spleen and kidney concentrations of the antibodyand any metabolites occurring 24 h after administration of theseconjugates. A more detailed description of the methods with respect toxenograft models can be found under C-6. Here, only the concentrationsof the metabolites of the conjugates according to the invention aredealt with. The values measured for the metabolites in the matricesmentioned additionally indicate how pronounced the metabolite burden isin plasma, kidney, spleen and liver, compared to the burden in thetumour.

Analysis for Quantification of the Potential Metabolites

The analysis of the compounds in the plasma, tumour, liver, spleen andkidney follows after precipitation of the proteins with generallymethanol by high-pressure liquid chromatography (HPLC) coupled to atriple-quadrupole mass spectrometer (MS).

For workup of 50 μl of plasma, 150 μl of precipitation reagent(generally methanol) are added and the mixture is shaken for 10 sec. Theprecipitation reagent contains an internal standard (ISTD) in a suitableconcentration (generally in the range of 20-100 μg/I). Aftercentrifugation at 1881 g for 10 minutes, the supernatant is transferredinto an autosampler vial, made up with 300 μl of a buffer matched to theeluent and shaken again.

In the workup of tumour or organ material, the particular material isadmixed with 3-20 times the amount of extraction buffer. The extractionbuffer contains 50 ml of Tissue Protein Extraction Reagent (Pierce,Rockford, IL), two pellets of Complete-Protease-Inhibitor-Cocktail(Roche Diagnostics GmbH, Mannheim, Germany) and phenylmethylsulfonylfluoride (Sigma, St. Louis, MO) in a final concentration of 1 mM.According to the tissue type (hard: tumour; soft: liver, kidney,spleen), the lysis and homogenization programme of the Prescellys 24lysis and homogenization system (Bertin Technologies) is selected(www.prescellys.com). The homogenized samples are left to stand at 4° C.overnight. 50 μl of the homogenizate are transferred into an autosamplervial and made up with 150 μl of methanol including ISTD, agitated for 10sec and then left to stand for 5 min. After adding 300 μl of ammoniumacetate buffer (pH 6.8) and agitating briefly, the sample is centrifugedat 1881 g for 10 minutes.

For calibration, plasma for plasma samples and corresponding blankmatrix for tissue samples is admixed with concentrations of 0.6-1000μg/l. According to the sample type or tissue type, the detection limit(LOQ) is between 1 and 20 μg/l.

The plasma and matrix samples are finally analysed using theHPLC-coupled API4200 triple-quadrupole mass spectrometer from AB SCIEXDeutschland GmbH.

Quality controls for testing validity contain 4, 40 and 400 μg/I.

Table 5: Concentrations of metabolite M1 in tumour, liver, kidney,spleen and plasma 24 h after single 10 mg/kg i.v. administration ofExample 1x-9024 in comparison to isotype control in REC-1 xenograft nunumice.

TABLE 5 Example M1 LLOQ Example Tissue Mean (μg/l) SD (μg/l) (μg/l)Isotype Tumour 54.2 0.7 20.0 control Liver 26.1 1.2 4.0 Kidney 93.3 9.310.0 Spleen 31.5 2.4 10.0 Plasma 4.2 0.8 1.0 1x-9024 Tumour 186.4 32.420.0 Liver 19.8 3.6 4.0 Kidney 28.0 7.0 10.0 Spleen 20.3 6.3 10.0 Plasma2.9 0.9 1.0

C-6 Activity Test In Vivo

The activity of the conjugates according to the invention can be testedin vivo, for example, using xenograft models. The person skilled in theart is familiar with methods in the prior art which allow the activityof the compounds according to the invention to be tested (see, forexample, WO 2005/081711; Poison et al., Cancer Res. 2009 Mar15;69(6):2358-64).

Human tumour cells which express the antigen for the antibody-drugconjugate are inoculated subcutaneously into the flank ofimmunosuppressed mice, for example NMRi nude or SCID mice. 1-10 millioncells are detached from the cell culture, centrifuged and resuspended inmedium or medium/matrigel. The cell suspension is injected under theskin of the mouse.

Within a few days, a tumour grows. Treatment is commenced after thetumour is established, at a tumour size of approximately 40 mm². Toexamine the effect on larger tumours, treatment may be initiated only ata tumour size of 50-100 mm².

Treatment with the ADCs is carried out via the intravenous (i.v.) routeinto the tail vein of the mouse. The ADC is administered in a volume of5 ml/kg.

The treatment protocol depends on the pharmacokinetics of the antibodyconjugate. Treatment was effected three times in succession everyseventh day as the standard. For a quick assessment, a protocol with asingle treatment may also be suitable. However, the treatment may alsobe continued, or a second cycle of further treatment days may follow atalater time.

As standard, 8 animals are used per treatment group. In addition to thegroups to which the active substances are administered, one group istreated as control group only with the buffer or isotonic salt solution,according to the same protocol.

During the experiment, the tumour area is measured regularly in twodimensions (length/width) using a caliper. The tumour area is determinedas length x width. The ratio of the mean tumour area of the treatmentgroup to that of the control group is stated as T/C area.

When, after the end of the treatment, all groups of the experiment areterminated at the same time, the tumours can be removed and weighed. Theratio of the mean tumour weights of the treatment group to that of thecontrol group is stated as T/C weight.

C-6a. Growth Inhibition/Regression of Experimental Tumours in the Mouse

The tumour cells (REC-1, MOLM-13 or MV-4-11) are inoculatedsubcutaneously into the flank of female NMRI-nude mice (Janvier). At atumour size of 40-50 mm², intravenous treatment is effected with theantibody-drug conjugate once a week for two or three weeks.

The treatment with the ADCs according to the invention leads to adistinct inhibition of tumour growth compared to the control group.Table 6 shows the T/C values determined for tumour area on therespective day of the end of the experiment, calculated from the startof treatment.

TABLE 6 Example Tumour model Dose Dose scheme T/C area R1x-9024 REC1 10mg/kg Q7dx3 0.48 (day 23) 1x-9024 0.19 (day 23) 1x-9024 REC1 10 mg/kgQ7dx3 0.22 (day 24) 1x-9574 0.20 (day 24) 1c-8988 MV-4-11  5 mg/kg Q7dx20.16 (day 20) 1c-9476 0.21 (day 20) 1c-8988 MOLM-13  5 mg/kg Q7dx2 0.33(day 14) 1c-9476 0.5 (day 14) 1c-8987 0.35 (day 14)

1. An antibody-drug conjugate of formula (I):

or a salt, solvate, or salt of a solvate thereof; wherein: n is 1-8, AKis an anti-CD123 antibody, or an anti-CXCRS antibody, or anantigen-binding fragment of these antibodies, wherein the antibody orthe antigen-binding fragment is attached via a sulfur atom of a cysteineside group.
 2. The antibody-drug conjugate of claim 1, wherein n is 2-8.3. The antibody-drug conjugate of claim 1 or 2, wherein n is 4-8.
 4. Apharmaceutical composition comprising at least one antibody-drugconjugate according to any one of claims 1 to 3, in combination with aninert, non-toxic, pharmaceutically suitable auxiliary.
 5. A method fortreating a disease in a subject comprising administering to the subjecta therapeutically effective amount of an antibody-drug conjugate of anyone of claims 1 to 3, or a salt, solvate, or salt of a solvate thereof.6. A method for treating a hyperproliferative and/or angiogenic disorderin a subject comprising administering to the subject a therapeuticallyeffective amount of an antibody-drug conjugate of any one of claims 1 to3, or a salt, solvate, or salt of a solvate thereof.
 7. A method fortreating cancer in a subject comprising administering to the subject atherapeutically effective amount of an antibody-drug conjugate of anyone of claims 1 to 3, or a salt, solvate, or salt of a solvate thereof.8. A method for treating cancer in a subject comprising administering tothe subject a therapeutically effective amount of an antibody-drugconjugate of any one of claims 1 to 3 in combination with one or moretherapeutic approaches for cancer immunotherapy or with one or moreactive compounds directed against a molecular target of cancerimmunotherapy.
 9. An anti-CD123 antibody comprising a variable region ofthe heavy chain (VH) comprising the variable CDR1 sequence of the heavychain (H-CDR1), as shown by SEQ ID NO: 12, the variable CDR2 sequence ofthe heavy chain (H CDR2), as shown by SEQ ID NO: 13 and the variableCDR3 sequence of the heavy chain (H-CDR3), as shown by SEQ ID NO: 14,and a variable region of the light chain (VL) comprising the variableCDR1 sequence of the light chain (L CDR1), as shown by SEQ ID NO: 16,the variable CDR2 sequence of the light chain (L-CDR2), as shown by SEQID NO: 17 and the variable CDR3 sequence of the light chain (L-CDR3), asshown by SEQ ID NO: 18, or an antigen-binding fragment thereof.
 10. Amethod for treating cancer in a subject comprising administering to thesubject a therapeutically effective amount of the anti-CD123 antibody ofclaim 9.